JP2024504167A - Antibody compositions and methods for treating hepatitis B virus infection - Google Patents

Abstract

The present disclosure relates to pharmaceutical compositions comprising antibodies that neutralize hepatitis B virus (HBV) infection. Furthermore, the present disclosure relates to the use of said pharmaceutical composition in the treatment of HBV infection.

Description

STATEMENT REGARDING THE SEQUENCE LISTING The sequence listing associated with this application is provided in text form in lieu of hard copy and is hereby incorporated by reference. The name of the text file containing the sequence listing is 930485_433WO_SEQUENCE_LISTING. txt. The text file is 124KB, was created on January 24, 2022, and will be submitted electronically via EFS-Web.

The present disclosure relates to pharmaceutical antibody compositions and methods for the prevention and treatment of hepatitis B virus infection.

HBV consists of (i) an envelope containing three associated surface proteins (hepatitis B surface antigen, HBsAg) and lipids, and (ii) an icosahedral nucleocapsid that encloses the viral DNA genome and DNA polymerase. . The HBV capsid is formed in the cytosol of infected cells during packaging of the RNA pregenome replication complex and gains the ability to bud during the synthesis of the viral DNA genome by reverse transcription of the pregenome in the particle lumen. The three HBV envelope proteins S-HBsAg, M-HBsAg, and L-HBsAg form complex transmembrane folds in the endoplasmic reticulum, forming disulfide-linked homo- and heterodimers. During budding at the intracellular membrane, a short linear domain of the cytosolic preS region interacts with binding sites on the capsid surface. Virions are then secreted into the blood. Additionally, surface proteins can bud in the absence of capsids and form subviral particles (SVPs) that are also secreted in 3-4 log excess over virions. High levels of HBsAg can deplete HBsAg-specific T cell responses and have been proposed as an important factor for viral immune tolerance in chronic hepatitis B (CHB) patients (Chisari FV, Isogawa M, Wieland SF, Pathologie Biologie, 2010;58:258-66).

Hepatitis B virus can cause fatal acute and chronic liver infections. Acute hepatitis B is characterized by viremia with or without symptoms, with a risk of developing fulminant hepatitis (Liang TJ, Block TM, McMahon BJ, Ghany MG, Guo JT, Locarnini S, Zoulim F, Chang KM, Lok AS. Present and future therapies of B hepatitis: From discovery to cure. Hepatology. 2015 Aug 3. doi: 10.1002/hep.28025. [Epub ahead of print]). Despite an effective vaccine against hepatitis B being available since 1982, the WHO estimates that 240 million people are chronically infected with hepatitis B, and more than 780,000 people die each year. It has been reported that patients died due to hepatitis B complications. Approximately one-third of patients with chronic hepatitis B (CHB) develop cirrhosis, liver failure, and hepatocellular carcinoma, accounting for 600,000 deaths per year (Liang TJ, Block TM, McMahon BJ, Ghany MG, Urban S, Guo JT, Locamini S, Zoulim F, Chang KM, Lok AS. Present and future therapies of hepatitis B: From discovery to cure. Hepatology. 2015 Aug 3. doi: 10.1002/hep.28025. [Epub ahead of print]).

For patients infected with HBV, severe complications may develop as a result of coinfection or superinfection with HDV. According to the WHO, approximately 15 million people worldwide are infected with hepatitis D. HDV is considered a subviral satellite because it can only be transmitted in the presence of HBV. HDV is one of the smallest known animal viruses (40 nm), resulting in a genome of only 1.6 kb and encodes S and L HDAg. All other proteins necessary for HDV genome replication, including RNA polymerase, are provided by the host cell and the HDV envelope is provided by HBV. Once introduced into a permissive cell, the HDV RNA genome replicates and associates with multiple copies of HDV-encoded proteins to assemble ribonucleoprotein (RNP) complexes. RNP is exported from the cell by HBV envelope proteins, which can also assemble lipoprotein vesicles that bud into the lumen of the pre-Golgi compartment before being secreted. Furthermore, HBV envelope proteins also provide a mechanism for targeting of HDV to uninfected cells, thereby ensuring spread of HDV.

Complications caused by HDV include an increased likelihood of experiencing rapid progression to liver failure and cirrhosis in acute infections and an increased likelihood of developing liver cancer in chronic infections. Hepatitis D in combination with hepatitis B virus has the highest mortality rate of all hepatitis infections at 20% (Fattovich G, Giustina G, Christensen E, Pantalena M, Zagni I, Realdi G, Schalm SW. Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. Gut. 2000 Mar;46(3):420-6). The only approved therapy for chronic HDV infection is interferon-alpha. However, treatment of HDV with interferon-alpha is relatively inefficient and poorly tolerated. Treatment with interferon-alpha results in a sustained virologic response 6 months after treatment in a quarter of patients. Nucleoside analogs (NAs) have also been widely tried in hepatitis delta, but appear to be ineffective. Combination treatment with NA and interferon also proved disappointing (Zaigham Abbas, Minaam Abbas Management of hepatitis delta: Need for novel therapeutic Options. World J Gastroenterol 2015 August 28; 21(32): 9461-9465) . Therefore, new treatment options are needed.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.

The figures provided herein are intended to more fully explain the subject matter contained in this disclosure. The figures in no way limit the disclosure. Throughout this disclosure, exemplary antibody HBC34v35 (with or without Fc mutations such as MLNS and GAALIE) is also referred to as HBC34-v35 and HBC34-V35. It will therefore be understood that HBC34v35, HBC34-v35 and HBC34-V35 have the same meaning. Similarly, exemplary antibody HBC34v34 is also referred to as HBC34-v34 and HBC34-V34, and exemplary antibody HBC34v7 is also referred to as HBC34-v7 and HBC34-V7. Furthermore, it will be understood that "MLNS-GAALIE" has the same meaning as "MLNS_GAALIE" (ie M428L+N434S+G236A+A330L+I332E mutation (EU numbering) in the Fc portion).

FIGS. 1A-1B depict HBsAg adw at the indicated concentrations of HBC34-v7 and two engineered antibodies of the present disclosure (“HBC34-v34”; “HBC34-v35”) as determined by a direct antigen-based ELISA assay. (1A) and HBsAg adr (1B). All antibodies were generated as IgG1 (g1m17, 1 allotype). Figures 2A-2K show the binding of HBC34-v7, HBC34-v34, and HBC34-v35 to all known HBsAg genotypes ((A)-(J), respectively) and mock controls (K). The genotype representative sequence representing the outer loop of the HBsAg antigen as shown in Example 5 of PCT Publication No. WO 2017/060504 was used. Staining was performed by FACS. Antibody concentration was as indicated on the y-axis of the graph. Figures 3A and 3B show binding of HBC34-v7 and HBC34-v35 containing wild-type or mutant Fc regions to HBsAg adw in a direct antigen-based ELISA assay (two experiments; data from "Experiment 1" Figure 3A and data from "Experiment 2" are shown in Figure 3B). Antigen binding curves are shown in the top panel of each figure. EC50 values (determined by fitting the curves using Graphpad Prism) are shown in the middle panel of each figure. Binding to uncoated plates (control) is shown in the bottom panel of each figure. Fc region: "HBC34v7" and "HBC34-v35" = wild type; "HBC34-v35-MLNS" = M428L/N434S. "HBC34-v35-MLNS-GAALIE" = M428L/N434S/G236A/A330L/I332E. Three lots of HBC34-v35 were tested. Two lots of HBC34-v35-MLNS and two lots of HBC34-v35-MLNS-GAALIE were tested. One lot of HBC34-v7 was used. Figures 4-7 show the effect of HBC34-v35 on serum HBAg levels in an in vivo mouse model of HBV infection. AAV/HBV infected SCID mice were transplanted with primary human hepatocytes and administered 1, 5, or 15 mg/kg of HBC34-v35, or PBS (control) as described in Example 5. Figure 4 shows serum HBV DNA concentrations before and after treatment. Figure 5 shows serum HBsAg concentrations before and after treatment. Figure 6 shows serum HBeAg concentrations before and after treatment. Figure 7 shows serum HBcrAg concentrations before and after treatment. Figures 8A-8E show binding of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE to human FcγRs as assessed by biolayer interferometry (BLI). 2 μg/ml of His-tagged human FcγR ((A) FcγRIIa allele H131; (B) FcγRIIa allele R131; (C) FcγRIIIa allele F158; (D) FcγRIIIa allele V158; (E) FcγRIIb) was Captured for 6 minutes with a PentaHis sensor. The FcγR-loaded sensor was then injected with 2 μg/ml of each mAb in the presence of 1 μg/ml of affiniPure F(ab') _2- fragment goat anti-human IgG, F(ab') ₂ -fragment specific (cross-linking human mAbs through Fab fragments). ml of kinetics buffer solution (pH 7.1) for 5 minutes (left part of the plot), followed by a dissociation step in the same buffer for an additional 4 minutes (right part of the plot). Binding and dissociation profiles were measured in real time as changes in interference patterns using Octet RED96 (ForteBio). Figure 9 shows binding of HBC34-v35-MLNS and HBC34-V35-MLNS-GAALIE to human C1q as measured by Octet. An anti-human Fab (CH1) sensor was used to capture intact IgG1 of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE mAbs at 10 μg/ml for 10 min through the Fab fragment. The IgG-loaded sensor was then exposed to a kinetics buffer solution (pH 7.1) containing 3 μg/ml of purified human C1q for 4 min (left part of the plot), followed by a dissociation step in the same buffer for an additional 4 min. (right part of plot). Binding and dissociation profiles were measured in real time as changes in interference patterns using Octet RED96 (ForteBio). Figures 10A and 10B show in vitro activation of human FcγRIIIa using NFAT-mediated receptor-bound activation of a luciferase reporter in engineered Jurkat cells. FcγRIIIa activation was tested using a validated commercially available bioreporter assay using recombinant HBsAg (Engerix B) as the target antigen. Serial dilutions of HBC34v35-MLNS and HBC34-v35-MLNS-GAALIE and control (Ctr) mAbs were incubated with HBsAg 0.2 μg/ml for 25 minutes at 37°C. Jurkat effector cells (Promega) expressing either the FcγRIIIa low affinity allele F158 (A) or the FcγRIIIa high affinity allele V158 (B) were resuspended in assay buffer and then added to the assay plate. After 24 hours of incubation at 37°C, Bio-Glo-™ Luciferase Assay Reagent (Promega) was added and luminescence was quantified using a luminometer (Bio-Tek). Figures 11A and 11B show in vitro activation of human FcγRIIa using NFAT-mediated receptor-bound activation of a luciferase reporter in engineered Jurkat cells. Activation of human FcγRIIa was tested using a validated commercially available bioreporter assay using recombinant HBsAg (Engerix B) as the target antigen. Serial dilutions of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE and control mAb (Ctr) were incubated with HBsAg 2 (A) or 0.2 μg/ml (B) for 25 min at 37°C. Jurkat effector cells (Promega) expressing the FcγRIIa high affinity allele H131 were resuspended in assay buffer and then added to the assay plate. After 23 hours of incubation at 37°C, Bio-Glo-™ Luciferase Assay Reagent (Promega) was added and luminescence was quantified using a luminometer (Bio-Tek). Figure 12 shows in vitro activation of human FcγRIIb using NFAT-mediated receptor-bound activation of a luciferase reporter in engineered Jurkat cells. Activation of human FcγRIIb was tested using a validated commercially available bioreporter assay using recombinant HBsAg (Engerix B) as the target antigen. Serial dilutions of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE and control mAb (Ctr) were incubated with HBsAg 1 μg/ml for 15 minutes at 37°C. Jurkat effector cells (Promega) expressing FcγRIIb were resuspended in assay buffer and then added to assay plates. After 20 hours of incubation at 37°C, Bio-Glo-™ Luciferase Assay Reagent (Promega) was added and luminescence was quantified using a luminometer (Bio-Tek). Figures 13A and 13B show in vitro killing of PLC/PRF/5 human hepatocellular carcinoma cells by human primary NK cells in the presence of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE. (A) ADCC was performed using freshly isolated NK cells from one donor previously genotyped for heterozygous high (V158) and low (F158) affinity FcγRIIIa (F/V) expression. Tested. Serial dilutions of HBC34-v35, HBC34-v35-MLNS, HBC34-v35-MLNS-GAALIE, 17.1.41, and control mAbs were added to the HBsAg-secreting hepatocellular carcinoma cell line PLC/PRF/5 (also called Alexander cells). ) was added. PLC/PRF/5 cells were incubated with antibodies for 10 minutes at room temperature. NK cells were added to the assay plate (10:1 effector to target cell ratio) and incubated for 4 hours at 37°C. Cell death was determined by measuring lactate dehydrogenase (LDH) release. (B) Staining of PLC/PRF/5 human hepatocellular carcinoma cells with HBC34v35 and 17.1.41 mAbs assessed by flow cytometry. Cells were extensively washed and fixed with formaldehyde (4%) or fixed and permeabilized (saponin 0.5%) before staining with various concentrations of HBC34-v35 and 17.1.41 mAb. Binding of these human mAbs was detected by flow cytometry using Alexa Fluor® 647 AffiniPure F(ab') ₂ fragment goat anti-human IgG, Fcγ fragment specific antibody. Figures 14A and 14B show in vitro activation of primary human NK cells in the presence of HBC34v35-MLNS and HBC34-v35-MLNS-GAALIE and HBsAg. NK cell activation was performed using freshly isolated cells from two donors previously genotyped for (A) homozygous high (V158) or (B) low (F158) affinity FcγRIIIa expression. I tested it. Serial dilutions of HBC34-V35, HBC34-v35-MLNS-GAALIE, and HBC34-v35-LALA mAbs were incubated with NK cells for 4 hours. NK cells were stained with anti-CD107a mAb as a functional marker to identify NK cell activity and NK cell activation was measured by flow cytometry. CD107a, also known as LAMP-1, is a marker for NK cell degranulation. 15A-15C depict healthy adult subjects in an exemplary single dose escalation (SAD) clinical trial of an exemplary pharmaceutical composition comprising antibody HBC34-v35-MLNS-GAALIE, as described in Example 9. The evaluation schedule is shown below. 16A-16E show the evaluation schedule for subjects with chronic HBV infection without cirrhosis and on nucleoside reverse transcriptase inhibitor (NRTI) therapy in the exemplary clinical trial described in Example 9. 17A-17C illustrate the time points of pharmacokinetic blood draws of subjects according to the exemplary clinical trial described in Example 9. FIG. 18 shows a dosing schedule from an exemplary clinical trial described in Example 9. FIG. 19 shows clinical laboratory evaluation according to the exemplary clinical study described in Example 9. FIG. 20 shows the activation and activation in monocyte-derived dendritic cells (moDCs) stimulated via immune complexes of HBC34-v35-MLNS+HBsAg; or HBC34-v35-MLNS-GAALIE+HBsAg, as described in Example 10. Showing upregulation of co-stimulatory markers. FIG. 21 shows secretion of cytokines by moDCs stimulated via immune complexes of HBC34-v35-MLNS+HBsAg; or HBC34-v35-MLNS-GAALIE+HBsAg, as described in Example 10. 22A and 22B in whole blood cultures stimulated via immune complexes containing HBC34-v35-MLNS and HBsAg; or HBC34-v35-MLNS-GAALIE and HBsAg, as described in Example 10. Showing the release of IFN-γ. Normalized (A) IFN-γ concentration (log10); (B) IFN-γ fold change (log10) as described in Example 10. 23A and 23B in whole blood cultures stimulated via immune complexes containing HBC34-v35-MLNS and HBsAg; or HBC34-v35-MLNS-GAALIE and HBsAg, as described in Example 10. Figure 2 shows the release of IL-2. Normalized (A) IL-2 concentration (log10); (B) IL-2 fold change (log10) as described in Example 10. 24A and 24B show whole blood cultures stimulated via immune complexes containing HBC34-v35-MLNS and HBsAg; or HBC34-v35-MLNS-GAALIE and HBsAg, as described in Example 10. IFN-γ and IL-2 in the figure are shown. (A) IFN-γ; 100 μg/ml mAb; (B) IL-2; IL-2 μg/ml mAb. FIG. 25 depicts an exemplary single dose escalation (SAD) cohort from the Phase 1 clinical trial described in Example 9 and Examples 11-16. FIG. 26 shows an in silico model for predicting a decrease in subject serum HBsAg after administration of HBC34-v35-MLNS-GAALIE 60 mg x 4 times. Figure 27 shows subjects who received HBC34-v35-MLNS-GAALIE at 6 mg (cohort 1b), subjects who received HBC34-v35-MLNS-GAALIE at 18 mg (cohort 2b), and HBC34-v35-MLNS at 75 mg. - Shows a table summarizing certain demographic and baseline characteristics of subjects who received GAALIE (cohort 3b). Figure 28 shows a table summarizing safety and tolerability data for Cohort 1b, Cohort 2b, and Cohort 3b. Figures 29A-29B show alanine aminotransferase (ALT) levels over time. (A) Shows ALT levels in cohort 1b (6 mg HBC34-v35-MLNS-GAALIE or placebo). (B) Shows ALT levels in cohort 2b (18 mg HBC34-v35-MLNS-GAALIE or placebo). Figures 30A-30B show HBC34-v35-MLNS-GAALIE 6mg x 1 dose S. C. or placebo (1b), HBC34-v35-MLNS-GAALIE 18 mg x 1 dose S. C. or placebo (2b), HBC34-v35-MLNS-GAALIE 75 mg x 1 dose S. C. or placebo (3b), HBC34-v35-MLNS-GAALIE 300 mg x 1 dose S. C. or placebo (4b), and HBC34-v35-MLNS-GAALIE 18 mg x 1 dose S. C. or an actual reduction in the level of HBsAg in the subject's serum after placebo (1c). (A) shows HBsAg change over time (Log 10 IU/mL). (B) shows absolute HBsAg (UI/mL) levels over time. Figure 31 shows HBC34-v35-MLNS-GAALIE 6mg x 1 dose S. C. or placebo, HBC34-v35-MLNS-GAALIE 18 mg x 1 dose S. C. or placebo, or HBC34-v35-MLNS-GAALIE 75m x 1 dose S. C. or the mean decrease in the level of HBsAg in the subject's serum after placebo. Data excludes participants with HBsAg decline <0.2 log ₁₀ IU/mL. Figure 32 shows HBC34-v35-MLNS-GAALIE 6mg x 1 dose S. C. or placebo, HBC34-v35-MLNS-GAALIE 18 mg x 1 dose S. C. or placebo, and HBC34-v35-MLNS-GAALIE 75 mg x 1 dose S. C. or detection of an HBsAg change from baseline in the level of HBsAg in a subject's serum after placebo. Figures 33A-33C show free PK profiles of HBC34-v35-MLNS-GAALIE in subject serum in cohorts 1b, 2b, 3b, and 1c. (A) shows free PK of HBC34-v35-MLNS-GAALIE in cohort 1b (6 mg). (B) shows free PK of HBC34-v35-MLNS-GAALIE in cohorts 2b (HBsAg<3000 IU/mL, HBeAg−) and 1c (18 mg, any HBsAg, HBeAg+/−). (C) shows free PK of HBC34-v35-MLNS-GAALIE in cohort 3b (75 mg). Figure 34 shows a summary of free PK parameters in cohorts 1b (6mg HBC34-v35-MLNS-GAALIE), 2b (18mg HBC34-v35-MLNS-GAALIE), and 3b (75mg HBC34-v35-MLNS-GAALIE). The data excludes one subject from 1b. Figures 35A-35C show the total PK profiles of HBC34-v35-MLNS-GAALIE in subject serum in cohorts 1b, 2b, 3b, and 1c. (A) shows the total PK of HBC34-v35-MLNS-GAALIE in cohort 1b (6 mg). (B) shows the total PK of HBC34-v35-MLNS-GAALIE in cohorts 2b (HBsAg<3000 IU/mL, HBeAg−) and 1c (18 mg, any HBsAg, HBeAg+/−). (C) shows the total PK of HBC34-v35-MLNS-GAALIE in cohort 3b (75 mg). Figure 36 shows Cohort 1b (6mg SC HBC34 -V35 -MLNS -GAALIE), 2B (18mg SC HBC34 -V35 -MLNS -GAALIE), 3B (75mg SC HBC34 -V35 -MLNS -GAALIE), and 1C (18mg SC). HBC34 -v35-MLNS-GAALIE). One subject was excluded from 1b. Figure 37 shows a table summarizing enrollment demographics for subjects in Part A of the trial. Including replacement subjects; BMI = body mass index. Figure 38 shows a table summarizing safety and tolerability data for subjects in Part A of the study. Includes alternate subjects; AE = adverse event; IV = intravenous; SC = subcutaneous. Figure 39 shows a table summarizing the serum pharmacokinetic parameters of HBC34-v35-MLNS-GAALIE after single subcutaneous or intravenous administration to healthy subjects. All parameters are shown as mean and %CV except t _1/2 , T _max , T _last which are shown as median (Q1, Q3). AUC = area under the curve; AUC _inf = AUC from time 0 to infinity; AUC _last = AUC to last measurable concentration; C _last = last measurable concentration; CL/F = apparent oral clearance; C _max = maximum concentration; IV = intravenous; SC = subcutaneous; T _last = time to last measurable concentration; T _max = time to reach C _max ; T _1/2 = half-life; V/F = apparent volume of distribution. . Figure 40 shows the serum concentration PK profile of HBC34-v35-MLNS-GAALIE after single subcutaneous or intravenous administration in healthy subjects. HV = healthy volunteer; PK = pharmacokinetics; IV = intravenous; SC = subcutaneous

The present disclosure provides pharmaceutical compositions comprising antibodies that neutralize hepatitis B virus (HBV) infection, and methods of using those compositions. In certain embodiments, the antibody binds HBsAg of a genotype selected from A, B, C, D, E, F, G, H, I, and J, or any combination thereof. In certain embodiments, the antibody has a mutation in the heavy chain that increases the in vivo half-life (e.g., in humans) of the antibody and binding affinity for an FcγR (e.g., human FcγRIIa, human FcγRIIIa, or both). Contains mutations in the heavy chain that improve sex.

In some embodiments, antibodies and pharmaceutical compositions are well tolerated by a subject when administered in a therapeutically effective amount. In some embodiments, the methods described herein include administering an antibody or pharmaceutical composition according to the present description to a subject infected with HBV.

Antibodies that neutralize HBV, pharmaceutical compositions comprising those antibodies, and methods for using such pharmaceutical compositions are described in detail below, but the specific methodologies described herein, It should be understood that this disclosure is not limited as protocols and reagents may vary. It should also be understood that the terms used herein do not limit the scope of the disclosure.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Below, aspects of the disclosure are described. Although certain specific embodiments are provided, it should be understood, however, that the embodiments of the present disclosure can be combined in any way and in any number to create further embodiments. The variously described examples and embodiments are not to be construed to limit the disclosure to the only explicitly described embodiments. This description should be understood to support and encompass embodiments that combine explicitly described embodiments with any disclosed subject matter. Furthermore, any permutations and combinations of all described subject matter in this application should be deemed to be disclosed by the description of this application, unless the context dictates otherwise.

Throughout this disclosure, unless the context otherwise requires, the term "comprise" and variations thereof, such as "comprises" and "comprising," are used, for example, "having," Used interchangeably with "has," "including," "includes," etc., and includes the listed components, ratios, whole numbers (including fractions thereof, where appropriate; e.g. 1/10 and 1/100), concentrations, or steps, but does not imply the exclusion of any other unlisted components, ratios, integers, concentrations, or steps. It will be. The term "consisting essentially of" is not equivalent to "comprising" and does not mean that the term "consisting essentially of" substantially affects the specified materials or steps of the claim or essential features of the claimed subject matter. Refers to materials or processes that do not provide For example, the amino acid sequences of a domain, region, module, or protein may, in combination, account for at most 20% (e.g., at most 15%, 10%, 8%, 6%) of the length of the domain, region, module, or protein. , 5%, 4%, 3%, 2% or 1%) and contributes to the activity of the domain(s), region(s), module(s), or protein (e.g., target binding of the binding protein). (i.e., does not reduce activity by more than 50%, e.g., 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% or less) , deletions, mutations, or combinations thereof (e.g., amino-terminal or carboxy-terminal or interdomain amino acids), a protein domain, region, or module (e.g., a binding domain) or a protein has a specific amino acid sequence. “becomes essentially from.”

The term "consist of" is a particular embodiment of the term "comprise" and excludes any other unlisted components, integers or steps. In the context of this disclosure, the term "comprising" encompasses the term "consisting of." The term "comprising" therefore includes "including" and "consisting." For example, a composition "comprising" X may consist exclusively of X, or may include something additional, eg, X+Y.

Furthermore, individual compounds or groups of compounds resulting from various combinations of structures and substituents described herein are referred to herein to the same extent as if each compound or group of compounds were individually described. should be understood as disclosed by. Therefore, the selection of particular structures or particular substituents is within the scope of this disclosure.

The terms "a" and "an" and "the" and similar references used in the context of describing this disclosure (including the context of the claims) are used herein unless otherwise indicated or outside of the context. Unless clearly contradicted, the terms should be construed to include both the singular and the plural. Use of an alternative word (eg, "or") should be understood to mean either one, both, or any combination of the alternative words. The recitation of ranges of values herein is intended to serve as a shorthand way of individually referring to each individual value within the range. Unless otherwise indicated herein, each individual value is incorporated into this disclosure as if individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the subject matter disclosed herein.

The word "substantially" does not exclude "completely"; for example, a composition "substantially free" of Y may be completely free of Y. In certain embodiments, "substantially" refers to a given amount of a composition, method, or use of the present disclosure compared to a given amount, effect, or activity of a reference composition, method, or use. , effect, or activity, and refers to 50% or less of the amount, effect, or activity of the reference composition, method, or use, such as 40%, 30%, 25%, 20%, 15%, 10%, 5%, or represents a decrease in amount, effect, or activity by 1% or less.

The term "about" with respect to a numerical value x refers to x±10%, such as x±5%, or x±7%, or x±10%, or x±12%, or x±15%, or x±20%. means. For example, in certain embodiments, "about" means ±20% of the indicated range, value, or structure.

"Optional" or "may, optionally" means that the element, component, event, or condition listed thereafter may or may not occur; A description is meant to include the occurrence and non-occurrence of an element, component, event, or condition.

The term "disease" as used herein refers to any of the following, all of which impair normal functioning, are typically manifested by characteristic signs and symptoms, and impair the longevity or quality of life of the afflicted human or animal. Generally synonymous with the terms "disorder" and "condition" (such as a medical condition) in that they reflect an abnormal condition of the human or animal body or one of its parts that results in a reduction in , are intended to be used interchangeably.

As used herein, the term "therapeutically effective" refers to the nature or amount of a pharmaceutical composition or antibody described herein that is sufficient to benefit a subject. In the context of this disclosure, the benefit also provided is the treatment of hepatitis B virus infection. As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, mitigation, recovery and therapy. Benefits of treatment include improved clinical outcome; reduction or alleviation of symptoms associated with the disease; decreased onset of symptoms; improved quality of life; longer disease-free state; decreased severity of disease; stabilization of disease status; Includes delaying disease progression; remission; survival; prolonging survival; or any combination thereof. The terms "subject" or "patient" are used interchangeably herein to mean a human susceptible to, or already infected with, HBV.

Doses are often expressed in relation to body weight (ie, of the subject). Thus, doses expressed as [g, mg, or other units]/kg (or g, mg, etc.) may be expressed as "kg (or g, mg, etc.)" even if the term "body weight" is not explicitly stated. etc.) per body weight [g, mg, or other units].

As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function like naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an alpha carbon attached to an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium refers to Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of amino acids, but that function similarly to naturally occurring amino acids.

As used herein, the terms "peptide," "polypeptide," and "protein," as well as variations of these terms, refer to at least two molecules linked to each other by a peptide bond (conventional or modified). Refers to molecules containing amino acids. For example, a peptide, polypeptide or protein may be composed of multiple amino acids selected from the 20 amino acids defined by the genetic code, each linked to at least one other amino acid by a peptide bond. A peptide, polypeptide or protein may be composed of L-amino acids and/or D-amino acids. The terms "peptide," "polypeptide," and "protein" are defined as peptide analogs that contain non-peptide structural elements and are defined as "peptide mimetics" in which the peptide can mimic or antagonize the biological effects of the natural parent peptide. Also includes the body. In certain embodiments, peptidomimetics lack characteristics such as enzymatically cleavable peptide bonds.

Peptides, polypeptides or proteins may contain, in addition to these amino acids, amino acids other than the 20 amino acids prescribed by the genetic code, or are composed of amino acids other than the 20 amino acids prescribed by the genetic code. Good too. In certain embodiments, peptides, polypeptides or proteins in the context of this disclosure undergo natural processes such as post-translational maturation processes, including those known in the art and described herein; It may also include amino acids that are modified by chemical processes (eg, synthetic processes). Such modifications may occur anywhere in the polypeptide, for example, in the peptide backbone, the amino acid chain, or at the carboxy or amino terminus. The peptide or polypeptide may be branched, such as after ubiquitination, or may be cyclic with or without branching. The terms "peptide," "polypeptide," and "protein" also include modified peptides, polypeptides, and proteins. For example, peptide, polypeptide or protein modifications include acetylation, acylation, ADP ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositol, Covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, Amino acid additions such as racemization, ceneroylation, sulfation, arginylation or ubiquitination may be included. Such modifications are described in the literature (Proteins Structure and Molecular Properties (1993) 2nd Ed., T. E. Creighton, New York; Post-translational Covalent Modifications of Proteins (1983) B. C. Johnson, Ed., Academic Press, New York; Seifter et al. (1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646 and Rattan et al., (1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663:48-62). Thus, the terms "peptide," "polypeptide," "protein" can include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like. Variants of the proteins, peptides, and polypeptides of this disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides have an amino acid sequence that is at least 70%, 75%, 80%, 85%, Comprising or consisting of amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical.

As used herein, "(poly)peptide" and "protein" may be used interchangeably with respect to a polymer of amino acid residues, such as multiple amino acid monomers linked by peptide bonds.

A "nucleic acid molecule" or "polynucleotide" or "nucleic acid" is a polymeric compound containing covalently linked nucleotides, which may be composed of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., a morpholine ring). refers to Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.

Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA) (including cDNA, genomic DNA, and synthetic DNA), any of which may be single-stranded or double-stranded. As long as it is single-stranded, the nucleic acid molecule may be a coding strand or a non-coding (antisense strand). Polynucleotides (including oligonucleotides), and fragments thereof, may be produced, for example, by polymerase chain reaction (PCR) or in vitro translation, or by ligation, cleavage, endonuclease action, or exonuclease action. good.

A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences encoding the same amino acid sequence. Some versions of the nucleotide sequence may also contain introns to the extent that introns can be removed through co-transcriptional or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as a result of redundancy or degeneracy of the genetic code, or by splicing, or both.

Variants of the nucleic acid molecules of the present disclosure are also contemplated. The variant nucleic acid molecule is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical, or 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C, or 0.015M sodium chloride, 0.0015M at about 42°C It hybridizes to polynucleotides under stringent hybridization conditions of sodium citrate and 50% formamide. A variant nucleic acid molecule retains the ability to encode a fusion protein or binding domain thereof that has the functionality described herein, such as specific binding to a target molecule.

As used herein, the term "sequence variant" refers to any sequence that has one or more changes compared to a reference sequence, which may include any published sequence and/or The "Table of Sequences and SEQ ID Numbers" (Sequence Listing), ie, the sequences listed in SEQ ID NO: 1 to SEQ ID NO: 120. Thus, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. In certain embodiments relating to sequence variants in the context of nucleotide sequences, the reference sequence is also a nucleotide sequence, whereas in certain embodiments relating to sequence variants in the context of amino acid sequences, the reference sequence is also an amino acid sequence. It is. "Sequence variant" as used herein means at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the reference sequence. , at least 96%, at least 97%, at least 98%, or at least 99% identical.

"Percent sequence identity" refers to the relationship between two or more sequences as determined by comparing the sequences. Methods for determining sequence identity can be designed to give the best match between the sequences being compared. For example, the sequences may be aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). . Furthermore, non-homologous sequences may be ignored for comparison purposes. Percent sequence identity referred to herein is calculated over the length of the reference sequence, unless otherwise indicated. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations can be performed using a BLAST program (eg, BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithms used in the BLAST program can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context of this disclosure, it will be understood that when sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the referenced program. "Default Values" means any set of values or parameters that are initially loaded into the software when it is first initialized.

A "sequence variant" in the context of a nucleic acid (nucleotide) sequence is one in which one or more nucleotides of the reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Has sequence variation. Nucleotides are referred to herein by their standard one-letter designation (A, C, G, or T). Due to the degeneracy of the genetic code, a "sequence variant" of a nucleotide sequence may or may not result in a change in the respective reference amino acid sequence, ie, an amino acid "sequence variant." In certain embodiments, the nucleotide sequence variant does not result in an amino acid sequence variant (eg, a silent mutation). In some embodiments, nucleotide sequence variants that result in "non-silent" mutations are contemplated. In some embodiments, the nucleotide sequence variants of the present disclosure are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% different from the reference amino acid sequence. %, at least 96%, at least 97%, at least 98%, or at least 99% identical. Nucleotide and amino acid sequences disclosed herein also refer to codon-optimized versions of reference or wild-type nucleotide or amino acid sequences. In any of the embodiments described herein, the polynucleotides of the present disclosure may be codon-optimized for the host cell containing the polynucleotide [e.g., Scholten et al., Clin. Immunol. 119: 135-145 (2006)].

A "sequence variant" in the context of an amino acid sequence has a sequence change in which one or more amino acids are deleted, substituted, or inserted compared to a reference amino acid sequence. As a result of the changes, such sequence variants are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% different from the reference amino acid sequence. %, at least 97%, at least 98%, or at least 99% identical. For example, a variant sequence having no more than 10 changes per 100 amino acids of the reference sequence, ie, any combination of deletions, insertions or substitutions, is "at least 90% identical" to the reference sequence.

A "conservative substitution" refers to an amino acid substitution that does not significantly affect or alter the binding properties of a particular protein. Generally, conservative substitutions are those in which the substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include those found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); 2: Aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K) , histidine (His or H); group 5: isoleucine (He or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and group 6: phenylalanine (Phe or F), Tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservatively substituted groups by similar function, chemical structure, or composition (eg, acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, the aliphatic class may include GIy, Ala, Val, Leu, and He for substitution purposes. Other conservative substitution groups include: sulfur-containing: Met and cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar positively charged residues: His, Arg, and Lys; large aliphatic nonpolar residues: Met, Leu, Ile , Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. It can be done. Examples of terminal insertions include fusion of the N-terminus or C-terminus of the amino acid sequence to a reporter molecule or enzyme.

Generally, changes in sequence variants do not eliminate or significantly reduce the desired functionality of the respective reference sequence. For example, the disclosed variant sequences bind to the same epitope and/or are more effective in inhibiting HBV and HDV infection than antibodies or antigen-binding fragments having (or encoded by) the reference sequences. It is preferred not to significantly reduce or completely suppress the functionality of the sequence of the antibody or antigen-binding fragment thereof to which it binds. Guidance in determining which nucleotide and amino acid residues, respectively, may be substituted, inserted or deleted without loss of the desired structure or functionality can be found using known computer programs. can.

As used herein, a nucleic acid or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide or protein refers to the origin of the nucleic acid, peptide, polypeptide or protein. A nucleic acid or amino acid sequence derived from a particular sequence may have an amino acid sequence that is essentially the same as that sequence or a portion thereof from which the nucleic acid or amino acid sequence is derived, whereby "essentially identical" does not mean . Including sequence variants as defined above. Nucleic acid or amino acid sequences derived from a particular peptide or protein may be derived from the corresponding domain of the particular peptide or protein. In this context, "corresponding" refers to possession of the same intended functionality or characteristics. For example, an "extracellular domain" corresponds to another "extracellular domain" (of another protein), or a "transmembrane domain" corresponds to another "transmembrane domain" (of another protein). . "Corresponding" portions of peptides, proteins and nucleic acids are therefore readily identifiable to those skilled in the art. Similarly, a sequence "derived from" another (eg, "source") sequence can be identified by one of skill in the art as having its origin in that source sequence.

A nucleic acid or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be identical to the starting nucleic acid, peptide, polypeptide or protein from which it is derived. However, a nucleic acid or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein also has one or more mutations compared to the starting nucleic acid, peptide, polypeptide or protein from which it is derived. In particular, a nucleic acid or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be a functional sequence as described above of the starting nucleic acid, peptide, polypeptide or protein from which it is derived. It may be a mutant type. For example, in a peptide/protein, one or more amino acid residues may be substituted with other amino acid residues, or one or more amino acid residue insertions or deletions may occur.

As used herein, the term "mutation" refers to a change in a nucleic acid and/or amino acid sequence as compared to a reference sequence, eg, a corresponding genomic sequence, wild-type sequence, or reference sequence. Mutations can be, for example, somatic mutations (naturally occurring), spontaneous mutations, induced mutations (e.g. induced by enzymes, chemicals or radiation), or site-specific mutations, e.g. compared to a reference genomic sequence. Mutations may also be obtained by specific mutagenesis, a molecular biological method for making specific and deliberate changes in nucleic acid and/or amino acid sequences. The term "mutation" or "mutating" shall therefore also be understood to include the physical creation of mutations, for example in a nucleic acid sequence or an amino acid sequence. Mutations include one or more nucleotide or amino acid substitutions, deletions and insertions, and several consecutive nucleotide or amino acid inversions. To achieve a mutation in an amino acid sequence, the nucleotide sequence encoding said amino acid sequence may be mutated to express a (recombinant) mutant polypeptide. Mutations can be made, for example, by changing a codon (e.g., changing one, two, or three nucleotide bases therein) of a nucleic acid molecule encoding a single amino acid (e.g., by site-directed mutagenesis). ), by providing codons encoding different or the same amino acids, or by synthesizing sequence variants.

The term "introduction" in the context of inserting a nucleic acid molecule into a cell means "transfection" or "transformation" or "transduction" and refers to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell. Nucleic acid molecules can be integrated into the genome of a cell (e.g., chromosomal, plasmid, plastid, or mitochondrial DNA), converted into autonomous replicons, or transiently expressed (e.g., transfected mRNA).

As used herein, the term "recombinant" (e.g., recombinant antibodies, recombinant proteins, recombinant nucleic acids, etc.) refers to those that are prepared, expressed, produced or isolated by recombinant means, and those that are naturally occurring. refers to any molecule (antibody, protein, nucleic acid, etc.) that does not exist in "Recombinant" can be used synonymously with "modified" or "non-naturally occurring" and refers to organisms, microorganisms, cells that contain at least one genetic change or that have been modified by the introduction of exogenous nucleic acid molecules; It may refer to a nucleic acid molecule, or a vector, in which such changes or modifications are introduced by genetic modification (ie, human intervention). Genetic modifications include, for example, modifications that introduce expressible nucleic acid molecules encoding proteins, fusion proteins or enzymes, or additions, deletions, substitutions of other nucleic acid molecules or other functional disruptions of the genetic material of a cell. It will be done. Additional modifications include, for example, non-coding regulatory regions where the modification alters expression of the polynucleotide, gene or operon.

As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to the host cell or subject; Refers to any gene, protein, compound, nucleic acid molecule, or activity that is naturally present in a host cell or subject that has been altered. Heterologous, non-endogenous, or exogenous is a gene, protein, compound, or nucleic acid molecule that has been mutated or otherwise differs in structure, activity, or both between the native and modified gene, protein, compound, or nucleic acid molecule. including genes, proteins, compounds, or nucleic acid molecules that have been altered. In certain embodiments, the heterologous, non-endogenous, or exogenous gene, protein, or nucleic acid molecule (e.g., receptor, ligand, etc.) may not be endogenous to the host cell or subject; Alternatively, the nucleic acid encoding such gene, protein, or nucleic acid molecule may be added to the host cell by conjugation, transformation, transfection, electroporation, etc., and the added nucleic acid molecule It may be integrated into the genome or it may exist as stained extragenetic material (eg, as a plasmid or other autonomously replicating vector). The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a naturally occurring polynucleotide or gene, and may encode a homologous polypeptide or activity; The structure, sequence, expression level, or any combination thereof may be altered. The non-endogenous polynucleotide or gene and the encoded polypeptide or activity may be derived from the same species, different species, or a combination thereof.

As used herein, the term "endogenous" or "naturally occurring" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or subject.

As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include primary subject cells and cultures derived therefrom, regardless of the number introduced. It is also understood that all progeny may not be exactly identical in DNA content due to deliberate or accidental mutations. Mutant progeny that have the same or substantially the same function, phenotype, or biological activity as screened for in the originally transformed cell are included. It will be clear from the context if a different name is intended.

The present disclosure is based in part on the design of antibodies and antigen-binding fragments that can neutralize hepatitis B and hepatitis delta viruses. Embodiments of antibodies and antigen-binding fragments according to the present description can be used in methods of preventing, treating, or attenuating HBV and HDV. In certain embodiments, the antibodies and antigen-binding fragments described herein have two or more different genotypes of the hepatitis B virus surface antigen and two or more different infectivity strains of the hepatitis B virus surface antigen. Binds to mutants. In certain embodiments, the antibodies and antigen-binding fragments described herein target all currently known genotypes of hepatitis B virus surface antigen, and all currently known genotypes of hepatitis B virus surface antigen. Binds all infectious mutants.

Antibodies and Antigen-Binding Fragments Thereof In one aspect, the present disclosure provides antibodies for binding to the antigenic loop region of HBsAg and for use in the pharmaceutical compositions and methods disclosed herein. Provided are isolated antibodies or antigen-binding fragments thereof that neutralize infection by.

As used herein, and unless the context clearly dictates otherwise, an "antibody" refers to at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. (although it is understood that heavy chain antibodies lacking light chains are also encompassed by the term "antibody"), as well as those having or retaining the ability to bind to the antigenic target molecule recognized by the intact antibody. refers to any antigen-binding portion or fragment of an intact antibody, such as a scFv, Fab, or F(ab')2 fragment. That is, the term "antibody" herein is used in its broadest sense and includes fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and polyclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including single chain antibody fragments, including single chain variable fragments (scFv) and single domain antibody (e.g. sdAb, sdFv, nanobody) fragments. and monoclonal antibodies. The term refers to genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, as well as heteroconjugate antibodies, multispecific, e.g. Includes bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless stated otherwise, the term "antibody" is understood to include functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass thereof, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

Thus, antibodies of the present disclosure may be of any isotype (eg, IgA, IgG, IgM, referred to as α, γ, and μ heavy chains, respectively). For example, in certain embodiments, the antibody is of the IgG type. Within the IgG isotype, antibodies may be of the IgG1, IgG2, IgG3, or IgG4 subclasses, such as IgG1. In some embodiments, the antibody comprises a constant region that includes amino acid sequences from two different isotypes (e.g., an exchange of the amino acid sequences of the constant domains), e.g., an amino acid sequence from an IgA antibody and an amino acid sequence from an IgG antibody. including. Antibodies of the present disclosure may include kappa or lambda light chains. In some embodiments, the antibody is of the IgG1 type and includes a kappa light chain.

As used herein, the terms "antigen-binding fragment,""fragment," and "antibody fragment" are used interchangeably, and any of the antibodies of the present disclosure that retains the antigen-binding activity of the antibody refers to a fragment of Examples of antibody fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab') ₂ , Fv, or scFv. Additionally, the term "antibody" as used herein includes both antibodies and antigen-binding fragments thereof. Antibodies and antigen-binding fragments are discussed further herein.

Human antibodies are known (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can be produced in transgenic animals (eg, mice) that, upon immunization, are capable of producing the complete repertoire of human antibodies, or a selection thereof, in the absence of endogenous immunoglobulin production. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies upon antigen stimulation (e.g. Jakobovits, A., et al., Proc. Natl. See Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 3340 ). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol. Biol. 222 (1991) 581-597). The methods of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P. , et al., J. Immunol. 147 (1991) 86-95). Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent Neutralization of SARS coronavirus. Nat Med. 10(8):871-5 may be prepared using improved EBV-B cell immortalization. The term "human antibody" as used herein also includes antibodies that have been modified, eg, in the variable regions, to produce the properties of the antibodies and antibody fragments of the present disclosure. As used herein, the term "variable region" [variable region of light chain (V _L ), variable region of heavy chain (V _H )] refers to the light chain and the variable region that are directly involved in the binding of the antibody to the antigen. Means each of the heavy chain pairs.

As used herein, the term "variable region" [e.g., light chain variable region (V _L ), heavy chain variable region (V _H )] refers to the light chain region of an antibody that is directly involved in the binding of the antibody to its antigen. Refers to the variable region of a chain or antibody heavy chain. In other words, the terms "V _L " or "VL" and "V _H " or "VH" refer to the variable binding region from the antibody light chain and antibody heavy chain, respectively.

The variable binding regions are made up of distinct subregions known as "complementarity determining regions" (CDRs) and "framework regions" (FRs). The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are generally used in the art to refer to the sequence of amino acids in an antibody variable region that confers antigen specificity and/or binding affinity. It is known to point to an array that does not. Generally, there are three CDRs in each variable region of an antibody. The VH and VL regions together include six CDRs: HCDR1, HCDR2, HCDR3, also referred to herein as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively; LCDR1, LCDR2, LCDR3. The CDRs on the heavy and/or light chains may be separated in the primary amino acid sequence by framework regions, such that framework regions (FRs) vary more than the CDRs [i.e., from antibody to antibody (e.g., the same allele (for each antibody encoded by)] is a region in the variable domain that is difficult to For example, a chain (or each chain, respectively) may consist of four framework regions separated by three CDRs. In certain embodiments, antibody VH comprises four FRs and three CDRs arranged as follows: FR1-CDRH1-FR2-CDHR2-FR3-CDHR3-FR4, and antibody VL comprises four FRs and three CDRs arranged as follows: FR1-CDRL1 - Contains 4 FRs and 3 CDRs, such as FR2-CDRL2-FR3-CDRL3-FR4. Generally, VH and VL together form an antigen binding site through their respective CDRs, but in some cases the binding site is formed by or includes one, two, three, four, or five CDRs. It will be understood that it is also possible to

As used herein, a "variant" of a CDR refers to a functional variant of a CDR sequence having one to three amino acid substitutions, deletions, or combinations thereof. Immunoglobulin sequences can be aligned to a numbering scheme (e.g., Kabat, EU, International Immunogenetics Information System (IMGT) and Aho), which is developed using the Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298 -300) can be used to annotate the positions of equivalent residues and allow comparisons for different molecules. It will be appreciated that in certain embodiments, antibodies or antigen-binding fragments of the present disclosure may include all or a portion of a heavy chain (HC), a light chain (LC), or both. For example, full-length, intact IgG antibody monomers typically include VH, CH1, CH2, CH3, VL, and CL. The Fc component is further described herein.

In this disclosure, CDR amino acid positions are defined according to the IMGT numbering system (IMGT: www.imgt.org/; see Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012). do.

Table 1 shows the heavy chain variable region (VH), light chain variable region (VL), CDR, heavy chain (HC), and light chain (LC) amino acid sequences of certain exemplary antibodies according to the present disclosure. .

Fragments of the antibodies described herein can be obtained from the antibodies by methods involving digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of antibodies can be obtained by cloning and expressing portions of the heavy or light chain sequences. Antibody "fragments" include Fab, Fab', F(ab')2, and Fv fragments. The present disclosure describes single chain Fv fragments (scFv), heavy or light chains derived from the heavy and light chains of the antibodies described herein, including, for example, scFvs comprising CDRs derived from antibodies according to the present invention. Also included are monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, and single chain antibodies in which the variable domains of the heavy and light chains are joined by a peptide linker.

In certain embodiments, antibodies or antigen-binding fragments thereof according to the present disclosure include purified antibodies, single chain antibodies, Fabs, Fab', F(ab')2, Fv, or scFv.

The antibodies and antigen-binding fragments of the present disclosure may be multispecific (i.e., bispecific, trispecific, tetraspecific, etc.) in embodiments, and any multispecific as disclosed herein. format. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are multispecific antibodies, such as bispecific or trispecific antibodies. Bispecific antibody formats are disclosed, for example, in Spiess et al., Mol. Immunol. 67(2):95 (2015), and Brinkmann and Kontermann, mAbs 9(2):182-212 (2017). , its bispecific format and methods of making it are incorporated herein by reference, such as bispecific T cell engagers (BiTEs), DART, Knobs-Into-Holes (KIH) assembly, scFv-CH3- KIH assembly, KIH common light chain antibody, TandAbs, triple body, TriBi minibody, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, intrabody, cross-Mab, dual Action Fab (DAF) (2-in-1 or 4-in-1), DutaMabs, DT-IgG, Charge-Pairs, Fab-Arm Exchange, SEED bodies, Triomabs, LUZ-Y assemblies, Fcabs, κλ bodies, Orthogonal Fabs, DVD- IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H) - IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (4 in 1). Bispecific or multispecific antibodies can be used in combination with another HBV and/or HDV-specific binding domain of the present disclosure, or with different HBV and/or HDV specific binding domains (e.g., at the same or different epitopes). HBV and/or HDV-specific binding domains of the present disclosure may be included in combination with binding domains or binding domains that specifically bind different antigens.

Antibody fragments of the present disclosure may undergo monovalent or multivalent interactions and may be included in a variety of structures as described above. For example, scFv molecules can be synthesized to create trivalent "triabodies" or tetravalent "tetrabodies." The scFv molecule may include an Fc region domain, resulting in a bivalent minibody. Additionally, the sequences of the present disclosure may be components of multispecific molecules in which the sequences of the present disclosure target other regions of the molecule where the sequences of the present disclosure bind to the epitopes of the present disclosure and other targets. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

In some embodiments, the antibody may be present in a pharmaceutical composition that is substantially free of other polypeptides, such as less than 90% (by weight) of the pharmaceutical composition, usually less than 60%, even usually less than 50%. less than 100% of the polypeptide consists of other polypeptides.

Antibodies according to the present disclosure may be immunogenic in human and/or non-human (or xenogeneic) hosts, such as mice. For example, an antibody may have an idiotope that is immunogenic in a non-human host but not in a human host. Antibodies of the present disclosure for human use include antibodies that are not typically isolated from mice, goats, rabbits, rats, non-primate mammals, other hosts, in some cases by humanization or Includes antibodies that cannot be obtained from xenogeneic mice. In certain embodiments, antibodies according to the present disclosure are non-immunogenic or substantially non-immunogenic in humans.

Variant forms of the disclosed antibodies that have been engineered to reduce known or potential immunogenicity and/or other possible disadvantages are also contemplated herein.

As used herein, a "neutralizing antibody" neutralizes, ie prevents, inhibits the ability of a pathogen to initiate and/or perpetuate an infection in a host (e.g., a host organism or host cell). , an antibody that can reduce, prevent, or interfere with this. The terms "neutralizing antibody," "neutralizing antibody," or "neutralizing antibody(s)" are used interchangeably herein. These antibodies may be used alone or in combination (e.g., two or more of the antibodies of the present disclosure, or antibodies of the present disclosure in antibody agents, including antibodies capable of neutralizing HBV B and/or D infection). (in combination with other agents, which may or may not be present), can be used as a prophylactic or therapeutic agent in a suitable formulation in conjunction with active vaccination.

As used herein, "specifically binds" or "specific for" refers to a specific binding interaction with an affinity or Ka (i.e., units of 1/M) greater than or equal to ^{10 5} M _{has an} equilibrium association constant of refers to the association or association of a non-binding protein (eg, an antibody or antigen-binding fragment thereof) or binding domain with a target molecule. Antibodies or binding domains can be classified as "high affinity" binding proteins or binding domains, or "low affinity" binding proteins or binding domains. A “high affinity” binding protein or binding domain is defined as at least 10 ⁷ M ⁻¹ , at least 10 ⁸ M ⁻¹ , at least 10 ⁹ M ⁻¹ , at least 10 ¹⁰ M ⁻¹ , at least 10 ¹¹ M ⁻¹ , at least 10 ¹² M ⁻¹ , or a binding protein or binding domain having a Ka of at least 10 ¹³ M ⁻¹ . A “low affinity” binding protein or binding domain refers to a binding protein or binding domain that has a Ka of up to 10 ⁷ M ⁻¹ , up to 10 ⁶ M ⁻¹ , or up to 10 ⁵ M ⁻¹ . Alternatively, affinity may be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (eg, 10 ⁻⁵ M to 10 ⁻¹³ M). The terms "binding" and "specifically binding" and similar references do not encompass non-specific adhesion.

In certain embodiments, antibodies according to the present disclosure are capable of binding to the antigenic loop region of HBsAg. The hepatitis B virus envelope generally consists of three "HBV envelope proteins" ("HBsAg", also known as "hepatitis B surface antigen"), namely the S protein (for "small" and also called S-HBsAg). ), M protein (represents “middle” and is also referred to as M-HBsAg), and L protein (represents “large” and is also referred to as L-HBsAg). S-HBsAg, M-HBsAg, and L-HBsAg share the same C-terminal tip (also referred to as the “S domain”; 226 amino acids), which corresponds to the S protein (S-HBsAg) and is responsible for the viral Important for assembly and infectivity. S-HBsAg, M-HBsAg, and L-HBsAg are synthesized in the endoplasmic reticulum (ER), assembled, and secreted as particles through the Golgi apparatus. The S domain contains four predicted transmembrane (TM) domains, which expose both the N-terminus as well as the C-terminus of the S domain to the lumen. Both transmembrane domains TM1 and TM2 are thought to be required for co-translational protein integration into the ER membrane, and transmembrane domains TM3 and TM4 are located at the C-terminal third of the S domain. The "antigenic loop region" of HBsAg is located between the predicted TM3 and TM4 transmembrane domains of the S domain of HBsAg, whereby the antigenic loop region comprises amino acids 101-172 of the S domain, which It contains 226 amino acids in total (Salisse J. and Sureau C., 2009, Journal of Virology 83: 9321-9328). The determinants of infectivity reside in the antigenic loop region of the HBV envelope protein. In particular, residues 119-125 of HBsAg contain a CXXC motif, which is thought to be important for HBV and HDV infectivity (Jaoude GA, Sureau C, Journal of Virology, 2005;79:10460- 6).

When a position in the amino acid sequence of the S domain of HBsAg is referred to herein, such position is referred to with respect to the amino acid sequence set forth in SEQ ID NO: 3 (shown below) or a natural or artificial sequence variant thereof. Created.
MENITSGFLGPLLVLVLQAGFLLTRILTRILTIPQSWTSWWTSWTSWWTSLGQNSPTSPTSPTSPTSPTSPTSPTCYRRFIFLLFILLCLVLLDY QGMLPVCPLIPGRTSTTTTSTTTSTTTSTTTTSTTTSTTTTTSTTTSTTTTTSTTTSTTTCRTC MttaqgtsmyPSCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVQWFVQWLSPTVIWMWGPSLYWGPSLYWGPSLYSLPSLPLLPLLPLLWVYI
(SEQ ID NO: 3, amino acids 101-172 are underlined)

For example, the expression "amino acids 101-172 of the S domain" refers to amino acid residues 101-172 of the polypeptide according to SEQ ID NO:3. However, one skilled in the art will appreciate that mutations or changes (including but not limited to substitutions, deletions, and/or additions, such as different genotypes of HBsAg or different HBsAg mutants described herein) It is understood that it can occur naturally in the amino acid sequence of the S domain of HBsAg or be artificially introduced into the amino acid sequence of the S domain of HBsAg without affecting the biological properties. Thus, as used herein, the term "S domain of HBsAg" encompasses all such polypeptides, including, for example, the polypeptide according to SEQ ID NO: 3 and natural or artificial mutants thereof. Furthermore, when sequence fragments of the S domain of HBsAg (e.g. amino acids 101-172 or amino acids 120-130 of the S domain of HBsAg) are described herein, these include not only the corresponding sequence fragment of SEQ ID NO: 3. , also the corresponding sequence fragments of its natural or artificial mutants. For example, the phrase "amino acid residues 101-172 of the S domain of HBsAg" includes the corresponding fragment of amino acid residues 101-172 of SEQ ID NO: 3 and mutants thereof (natural or artificial). do. As used herein, the phrases "corresponding sequence fragments" and "corresponding fragments" refer to sequences that have been subjected to an optimized alignment, i.e., such that the sequences are aligned such that the maximum percentage identity is obtained. Refers to the fragments that are located at the same position in the array when they are aligned.

The M protein (M-HBsAg) corresponds to the S protein extended by a 55 amino acid N-terminal domain called "pre-S2". The L protein (L-HBsAg) corresponds to the M protein extended by a 108 amino acid N-terminal domain called "pre-S1" (genotype D). The pre-S1 and pre-S2 domains of the L protein are thought to be present on the inner surface of the virus particle (cytoplasmic side of the ER) and play an important role in virus assembly, or on the outer surface (in the lumen of the ER). side) and is used for interaction with target cells and is important for viral infectivity. Furthermore, HBV surface proteins (HbsAg) are not only incorporated into the virion envelope but also spontaneously bud from the ER-Golgi intermediate compartment membrane and form empty "subviral particles" (SVPs) that are released from the cell by secretion. can be formed.

In some embodiments, the antibody or antigen-binding fragment binds to the antigenic loop region of HBsAg and is capable of binding all of S-HBsAg, M-HBsAg, and L-HbsAg.

In some embodiments, the antibody or antigen-binding fragment neutralizes infection by hepatitis B virus and hepatitis delta virus. In some embodiments, the antibody or antigen-binding fragment reduces viral infectivity of hepatitis B virus and hepatitis delta virus.

To study and quantify viral infectivity (or "neutralization") in the laboratory, standard "neutralization assays" may be utilized. For neutralization assays, animal viruses are typically grown in cells and/or cell lines. Neutralization assays may be used in which cultured cells are incubated with a fixed amount of HBV or HDV in the presence (or absence) of the antibody (or antigen-binding fragment) to be tested. In such assays, levels of hepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant are used, and/or staining of HBcAg is assessed, A readout is obtained. For example, for HDV, delta antigen immunofluorescence staining may be evaluated.

In certain embodiments of HBV neutralization assays, cultured cells, eg, HepaRG cells, such as differentiated HepaRG cells, are incubated with an amount of HBV in the presence or absence of the antibody to be tested. In such embodiments, incubation is performed for 16 hours at 37°C, for example. Incubation may be carried out in medium (eg supplemented with 4% PEG 8000). After incubation, cells are washed and further cultured. To measure the infectivity of the virus, for example, the levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted into the culture supernatant from day 7 to day 11 after infection are It can be determined by enzyme-linked immunosorbent assay (ELISA). Additionally, HBcAg staining can be evaluated in an immunofluorescence assay. In HDV neutralization assay embodiments, substantially the same assay as for HBV may be used, with the difference that serum from an HDV carrier may be used (instead of HBV) as the HDV infection inoculum to differentiated HepaRG cells. There is. For detection, delta antigen immunofluorescence staining can be used as a readout.

Embodiments of antibodies of the present disclosure have high neutralizing capacity (eg, in vitro). For example, in certain embodiments, the concentration of an antibody described herein required for 50% neutralization of hepatitis B virus (HBV) or hepatitis delta virus (HDV) is, for example, about 10 μg/ml or less. . In other embodiments, the concentration of antibody required for 50% neutralization of HBV and HDV is about 5 μg/ml. In other embodiments, the concentration of antibodies described herein required for 50% neutralization of HBV and HDV is about 1 μg/ml. In yet other embodiments, the concentration of antibody required for 50% neutralization of HBV and HDV is about 750 ng/ml. In further embodiments, the concentration of antibodies described herein required for 50% neutralization (eg, in vitro) of HBV and HDV is 500 ng/ml or less. In such embodiments, the concentration of antibodies described herein required for 50% neutralization of HBV and HDV is 450 ng/ml or less, 400 ng/ml or less, 350 ng/ml or less, 300 ng/ml or less, 250 ng/ml or less. ml or less, 200ng/ml or less, 175ng/ml or less, 150ng/ml or less, 125ng/ml or less, 100ng/ml or less, 90ng/ml or less, 80ng/ml or less, 70ng/ml or less, 60ng/ml or less, or 50ng /ml or less.

Antibodies or antigen-binding fragments according to the present disclosure that are capable of neutralizing both HBV and HDV are useful in the prevention and treatment of hepatitis B and hepatitis D. Infection with HDV typically occurs simultaneously with or following infection with HBV (e.g., HDV requires HBV support for its own replication, so inoculation with HDV in the absence of HBV does not cause hepatitis D infection. Hepatitis D is typically observed in chronic HBV carriers.

Embodiments of the disclosed antibodies promote clearance of HBsAg and HBV. In certain embodiments, the antibody promotes clearance of both HBV and hepatitis B virus subviral particles (SVP). Elimination of HBsAg or subviral particles may be assessed, for example, by measuring the level of HBsAg in, for example, a blood sample of a hepatitis B patient. Similarly, clearance of HBV can be assessed, for example, by measuring the level of HBV in, for example, a blood sample of a hepatitis B patient.

In addition to infectious particles (HBV), the serum of patients infected with HBV contains an excess of HBV envelope protein (HBsAg), typically in the form of relatively small spheres and fibers of variable length. There are (typically 1,000 to 100,000 times) empty subviral particles (SVPs). Subviral particles have been shown to strongly enhance intracellular viral replication and HBV gene expression (Bruns M. et al. 1998 J Virol 72(2): 1462-1468). This is also relevant in the context of the infectivity of HBV-containing sera, since infectivity depends not only on the number of viruses but also on the number of SVPs (Bruns M. et al. 1998 J Virol 72(2): 1462- 1468). Additionally, excess subviral particles may act as decoys by absorbing neutralizing antibodies, thus delaying clearance of infection. Achieving loss of hepatitis B surface antigen (HBsAg) is considered in some cases to be the end point of treatment and the proximate outcome of curing chronic hepatitis B (CHB).

Embodiments of antibodies of the present disclosure may promote clearance of HBsAg. In certain embodiments, the antibody promotes clearance of subviral particles of hepatitis B virus. In some embodiments, antibodies can be used (eg, in pharmaceutical compositions of the present disclosure) to treat chronic hepatitis B.

In any of the embodiments of the present disclosure, the antibody or antigen-binding fragment is selected from HBsAg genotypes A, B, C, D, E, F, G, H, I, and J or any combination thereof. binds to HBsAg of the given genotype.

In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are of HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. , 5, 6, 7, 8, 9, or 10. Examples of various HBsAg genotypes include: GenBank accession number J02203 (HBV-D, ayw3); GenBank accession number FJ899792.1 (HBV-D, adw2); GenBank accession number AM282986 (HBV-A); Accession number D23678 (HBV-B1 Japan); GenBank accession number AB117758 (HBV-C1 Cambodia); GenBank accession number AB205192 (HBV-E Ghana); GenBank accession number X69798 (HBV-F4 Brazil) ); GenBank accession number AF160501 (HBV- GenBank accession number AY090454 (HBV-H Nicaragua); GenBank accession number AF241409 (HBV-I Vietnam); and GenBank accession number AB486012 (HBV-J Borneo). Exemplary amino acid sequences of the antigenic loop region of the S domain of HBsAg of various genotypes are described herein (eg, SEQ ID NOs: 5-15).

In some embodiments, the antibody or antigen-binding fragment binds at least 6 of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In certain embodiments, the antibody or antigen-binding fragment binds at least 8 of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In some embodiments, the antibody or antigen-binding fragment binds all ten of the ten HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. HBV is differentiated into several genotypes according to the genome sequence. To date, eight well-known genotypes (A-H) of the HBV genome have been defined. Furthermore, two other genotypes, namely I and J, have also been identified (Sunbul M., 2014, World J Gastroenterol 20(18): 5427-5434). Genotype is known to influence disease progression, and differences between genotypes in response to antiviral treatment have been determined.

In some embodiments, antibodies or antigen-binding fragments according to the present disclosure are HBsAg mutants having mutations in the antigenic loop region. , 11, 12, 13, 14, 15, 16, 17, or 18, such mutants include HBsAg Y100C/P120T, HBsAg P120T, HBsAg P120T/S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBs selected from one or more of Ag G145R, and HBsAg N146A. These mutants are naturally occurring mutants based on the S domain (SEQ ID NO: 4) of HBsAg genotype D, GenBank accession number FJ899792. The mutated amino acid residue in each of the mutants noted herein is indicated by its name.

Sequence number 4
MenvtsgflgPLLVLQAGFFLLTRILTRILTIPQSWWTSWWTSWWTSWTSLGQNSPTSNSPTSPTSPTSPTCYRFIFLLFILLCLFLLCLVLDY QGMLPVCPLIPLIPCRTCTC TTPAQGTSMYPSCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVQWFVQWLSPTVIWMWGPSLYWGPSLPSLPLLPLLPLLWVYI
(Antigenic loop region, i.e. amino acids 101-172 is underlined)

The amino acid sequences of the antigenic loop region of the S domain of various mutant HBsAg are shown in SEQ ID NOs: 16-33.

In certain embodiments, the antibody or antigen-binding fragment is HBsAg Y100C/P120T, HBsAg P120T, HBsAg P120T/S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg Binds at least 12 infectious HBsAg mutants selected from M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A. In some such embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure are HBsAg Y100C/P120T, HBsAg P120T, HBsAg P120T/S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q 129H, selected from HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A Binds at least 15 infectious HBsAg mutants. In some embodiments, the antibody or antigen-binding fragment is directed against the following infectious HBsAg mutants: HBsAg Y100C/P120T; HBsAg P120T; HBsAg P120T/S143L; HBsAg C121S; HBsAg R122D; HBsAg R122I; HBsAg T123N; sAg Q129H; HBsAg Q129L; HBsAg M133H; HBsAg M133L; HBsAg M133T; HBsAg K141E; HBsAg P142S; HBsAg S143K; HBsAg D144A; HBsAg G145R; and HBsAg N1 46A.

In certain embodiments, the antibody or pharmaceutical composition comprising the same reduces serum concentrations of HBV DNA in a mammal with an HBV infection. In certain embodiments, the antibody or pharmaceutical composition comprising the same reduces serum levels of HBsAg in a mammal with an HBV infection. In certain embodiments, the antibody or pharmaceutical composition comprising the same reduces serum concentrations of HBeAg in a mammal with an HBV infection. In certain embodiments, the antibody or pharmaceutical composition comprising the same reduces serum levels of HBcrAg in a mammal with an HBV infection.

The term "epitope" or "antigenic epitope" refers to any molecule that is recognized and specifically bound by an associated binding molecule, such as an immunoglobulin, chimeric antigen receptor, or other binding molecule, domain, or protein; including structure, amino acid sequence, or protein determinants. Epitopic determinants generally include chemically active surface groupings of molecules such as amino acids or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics.

In some embodiments, the antibody or antigen-binding fragment binds to an epitope comprising at least one, at least two, at least three, or at least four amino acids of the antigenic loop region of HBsAg. In certain embodiments, the antibody or antigen-binding fragment has at least one selected from amino acids 115-133 of the S domain of HBsAg, amino acids 120-133 of the S domain of HBsAg, or amino acids 120-130 of the S domain of HBsAg. Combines with two amino acids. In certain embodiments, the antibody or antigen-binding fragment has at least one selected from amino acids 115-133 of the S domain of HBsAg, amino acids 120-133 of the S domain of HBsAg, or amino acids 120-130 of the S domain of HBsAg. Combines with three amino acids. In some embodiments, the antibody or antigen-binding fragment is at least selected from amino acids 115-133 of the S domain of HBsAg, amino acids 120-133 of the S domain of HBsAg, or amino acids 120-130 of the S domain of HBsAg. Combines with 4 amino acids. As used herein, amino acid positions (e.g., 115-133, 120-133, 120-130) are defined in all three HBV envelope proteins S-HBsAg, M-HBsAg, and L-HBsAg. Refers to the S domain of the above-mentioned HBsAg present, whereby S-HBsAg typically corresponds to the S domain of HBsAg.

The term "formed by" as used herein in the context of an epitope indicates that the epitope to which the antibody or antigen-binding fragment thereof binds may be linear (contiguous) or conformational (discontinuous). means. A linear or sequential epitope is an epitope that is recognized by an antibody according to its linear sequence of amino acids or primary structure. Conformational epitopes are recognized according to their three-dimensional shape and structure of the protein. Therefore, if an epitope is a linear epitope and contains two or more amino acids located at positions selected from amino acids 115-133 or amino acids 120-133 of the S domain of HBsAg, Amino acids may be located in contiguous locations in the primary structure (eg, consecutive amino acids in an amino acid sequence). In the case of a conformational epitope (3D structure), the amino acid sequence typically forms a 3D structure as an epitope, so the amino acids forming the epitope may or may not be located adjacent to each other in the primary structure. (ie, they may or may not be consecutive amino acids in the amino acid sequence).

In certain embodiments, the epitope to which the antibody or antigen-binding fragment binds is a conformational epitope. In some embodiments, the antibody or antigen-binding fragment binds an epitope comprising at least two amino acids of the antigenic loop region of HBsAg, wherein said at least two amino acids are amino acids 120-133 of the S domain of HBsAg or amino acids 120-130, said at least two amino acids are not located in adjacent positions (of the primary structure). In certain embodiments, the antibody or antigen-binding fragment binds an epitope comprising at least three amino acids of the antigenic loop region of HBsAg, and said at least three amino acids are amino acids 120-133 of the S domain of HBsAg or amino acids 120-130, and at least two of said three amino acids are not located in adjacent positions (of the primary structure). In some embodiments, the binding protein binds an epitope comprising at least 4 amino acids of the antigenic loop region of HBsAg, and the at least 4 amino acids are selected from amino acids 120-133 or amino acids 120-130 of the S domain of HBsAg. and at least two of the four amino acids are not located in adjacent positions (in the primary structure).

Amino acids that are not located adjacent to the primary structure and to which an antibody or antigen-binding fragment of the present disclosure binds (i.e., amino acids that form an epitope) may in some cases separated by one or more non-bonding amino acids. In some embodiments, at least 1, at least 2, at least 3, at least 4, or at least 5 amino acids are located between two non-adjacently located amino acids included in the epitope. You can leave it there.

In certain embodiments, the antibody or antigen-binding fragment binds to an epitope comprising at least amino acids P120, C121, R122, and C124 of the S domain of HBsAg. In other embodiments, the antibodies or antigen-binding fragments of the present disclosure have SEQ ID NO: 88:
PCRXC
binds to an epitope comprising an amino acid sequence, where X is any amino acid or no amino acid, X is any amino acid, is T, Y, or R, or X is T or R.

In other embodiments, the antibodies or antigen-binding fragments of the present disclosure have SEQ ID NO: 80:
T.G.P.C.R.T.C.
or an amino acid sequence that shares at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:80.

In other embodiments, the antibody or antigen-binding fragment of the present disclosure has SEQ ID NO: 85:
STTSTG PCRTC
or an amino acid sequence that shares at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:85.

In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure bind at least amino acids 145-151 of the S domain of HBsAg.
GNCTCIP
(Sequence number 81)
Binds to an epitope containing an amino acid sequence containing.

In yet other embodiments, the antibodies or antigen-binding fragments of the present disclosure bind to an epitope comprising the amino acid sequence according to SEQ ID NO: 80 and the amino acid sequence according to SEQ ID NO: 81.

In other embodiments, the antibodies or antigen-binding fragments of the present disclosure bind to an epitope comprising the amino acid sequence according to SEQ ID NO: 85 and/or the amino acid sequence according to SEQ ID NO: 87.

As mentioned above, the epitope to which an antibody or antigen-binding fragment of the present disclosure binds may be linear (contiguous) or conformational (discontinuous). In some embodiments, an antibody or antigen-binding fragment of the present disclosure binds a conformational epitope, and in certain such embodiments, the conformational epitope is present only under non-reducing conditions.

In certain embodiments, antibodies or antigen-binding fragments of the present disclosure bind linear epitopes. In certain such embodiments, the linear epitope is present under both non-reducing and reducing conditions.

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure has SEQ ID NO: 1:
X ₁ X ₂ X ₃ TCX ₄ X ₅ X ₆ AX ₇ G
binds to an epitope in the antigenic loop of HBsAg formed by the amino acid sequence where _X1 , _X2 , _X3 , _X4 , _X5 , _X6 , and _X7 can be any amino acids. (SEQ ID NO: 1).

In some embodiments, _X1 , _X2 , _X3 , _X4 , _X5 , _X6 , and _X7 are conservatively substituted amino acids compared to amino acids 120-130 of SEQ ID NO:3. . In some embodiments, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X _{6 ,} and It is an amino acid.

In certain embodiments, amino acid X1 of SEQ ID NO: ₁ is a small amino acid. A "small" amino acid, as used herein, refers to any amino acid selected from the group consisting of alanine, aspartic acid, asparagine, cysteine, glycine, proline, serine, threonine, and valine. In certain such embodiments, X ₁ is proline, serine, or threonine.

In certain embodiments, _X2 of SEQ ID NO: 1 is a small amino acid. In certain embodiments, X ₂ may be selected from cysteine or threonine.

In some embodiments, X ₃ of SEQ ID NO: 1 is a charged or aliphatic amino acid. "Charged" amino acid, as used herein, refers to any amino acid selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid, and histidine. "Aliphatic" amino acid, as used herein, refers to any amino acid selected from the group consisting of alanine, glycine, isoleucine, leucine, and valine. In certain embodiments, X ₃ is selected from arginine, lysine, aspartic acid, or isoleucine.

In some embodiments, X ₄ of SEQ ID NO: 1 is a small amino acid and/or a hydrophobic amino acid. A "hydrophobic" amino acid, as used herein, refers to any amino acid selected from the group consisting of alanine, isoleucine, leucine, phenylalanine, valine, tryptophan, tyrosine, methionine, proline, and glycine. In certain embodiments, X ₄ is selected from methionine or threonine.

In some embodiments, _X5 of SEQ ID NO: 1 is a small amino acid and/or a hydrophobic amino acid. In certain embodiments, X ₅ is selected from threonine, alanine, or isoleucine.

In some embodiments, X ₆ of SEQ ID NO: 1 is a small amino acid and/or a hydrophobic amino acid. In certain embodiments, X ₆ is selected from threonine, proline, or leucine.

In some embodiments, _X7 of SEQ ID NO: 1 is a polar or aliphatic amino acid. "Polar" amino acid, as used herein, refers to any amino acid selected from the group consisting of aspartic acid, asparagine, arginine, glutamic acid, histidine, lysine, glutamine, tryptophan, tyrosine, serine, and threonine. . In certain embodiments, _X7 is glutamine, histidine, or leucine.

In some embodiments, a binding protein according to the present disclosure has SEQ ID NO: 2:
X ₁ X ₂ X ₃ TCX ₄ X ₅ X ₆ AX ₇ G
binds to an epitope in the antigenic loop of HBsAg formed by the amino acid sequence where X ₁ is P, T, or S,
_X2 is C or S,
X ₃ is R, K, D, or I,
X ₄ is M or T,
X ₅ is T, A, or I,
X ₆ is T, P, or L, and X ₇ is Q, H, or L.
(Sequence number 2)

With respect to an epitope formed by an amino acid sequence according to SEQ ID NO: 1 or 2, the term "formed by" as used herein means that the disclosed binding protein binds necessarily every amino acid of SEQ ID NO: 1 or 2. It should be noted that this does not imply that In particular, the binding protein may bind only some of the amino acids of SEQ ID NO: 1 or 2, other amino acid residues may act as "spacers".

In certain embodiments, antibodies or antigen-binding fragments according to the present disclosure have one or more, two or more, three or more, or four amino acid sequences selected from SEQ ID NOs: 5-33 set forth in Table 3 below. It binds to an epitope in the antigenic loop of HBsAg formed by the above amino acids.

In some embodiments, an antibody or antigen-binding fragment according to the present disclosure has an antigenic loop region of HBsAg or a sequence thereof having an amino acid sequence according to any one or more of SEQ ID NOs: 5-33 set forth in Table 3 below. Binds to mutants. In certain embodiments, antibodies or antigen-binding fragments according to the present disclosure bind all antigenic loop variants of HBsAg having an amino acid sequence according to any of SEQ ID NOs: 5-33 set forth in Table 3 below.

Table 3: Exemplary amino acid sequences of the antigenic loop region of the S domain of HBsAg of various genotypes and mutants used herein (residues 101-172 of the S domain of HBsAg, including associated mutations). (excluding SEQ ID NO: 16, which refers to residues 100 to 172 of the S domain of HBsAg).

Thus, in certain aspects, the present disclosure provides for (i) a heavy chain variable region (V _H ) comprising at least 90% identity with an amino acid sequence according to SEQ ID NO: 41 or 67; , 89, 90, or _110-120 , suitable for use in the pharmaceutical compositions and methods of the present disclosure. Provided is an isolated antibody or antigen-binding fragment thereof, provided that the amino acid at position 40 of VL according to IMGT numbering is not cysteine, and the antibody or antigen-binding fragment thereof binds to the antigenic loop region of HBsAg to form B. Neutralizes infections caused by hepatitis virus and hepatitis delta virus.

In further embodiments, (i) the V _H comprises at least 95% identity with the amino acid sequence according to SEQ ID NO: 41 or 67, and/or (ii) the V _L comprises SEQ ID NO: 42, 59, 65, 89, 90, or at least 95% identity with an amino acid sequence according to any one of 110-120.

In certain embodiments, the amino acid at position 40 of the V _L is alanine. In other embodiments, the amino acid at position 40 of the V _L is serine. In yet other embodiments, the amino acid at position 40 of the V _L is glycine.

In any of the embodiments disclosed herein, antibodies or antigen-binding fragments suitable for use in the pharmaceutical compositions and methods of the present disclosure include SEQ ID NO: (i) 34-36, 37, 38, respectively; and 40; (ii) 34, 66, 36, 37, 38, and 40, respectively; (iii) 34-36, 37, 39, and 40, respectively; (iv) 34, 66, 36, 37, 39, and 40; (v) 34-36, 37, 38, and 58, respectively; (vi) 34, 66, 36, 37, 38, and 58, respectively; (vii) 34-36, 37, 39, and 58, respectively; or (vii) may include CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences according to 34, 66, 36, 37, 39, and 58;

In some embodiments, the V _L of an antibody or antigen-binding fragment suitable for use in the pharmaceutical compositions and methods of this disclosure comprises or consists of the amino acid sequence according to SEQ ID NO:89. In some embodiments, the V _L of an antibody or antigen-binding fragment suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises or consists of the amino acid sequence according to SEQ ID NO:90. In other embodiments, the V _L of an antibody or antigen-binding fragment suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 109-120. Become. In certain embodiments, the V _H comprises or consists of the amino acid sequence according to SEQ ID NO:41. In other embodiments, the V _H comprises or consists of the amino acid sequence according to SEQ ID NO: 67.

In certain embodiments, the V _H comprises or consists of the amino acid sequence according to SEQ ID NO: 41 and the V _L comprises or consists of the amino acid sequence according to SEQ ID NO: 89. In other embodiments, the V _H comprises or consists of the amino acid sequence according to SEQ ID NO: 41 and the V _L comprises or consists of the amino acid sequence according to SEQ ID NO: 90. In certain embodiments, the V _H comprises or consists of the amino acid sequence according to SEQ ID NO: 41 and the V _L comprises or consists of the amino acid sequence according to any one of SEQ ID NO: 109-120. In other embodiments, the V _H comprises or consists of the amino acid sequence according to SEQ ID NO: 67 and the V _L comprises or consists of the amino acid sequence according to any one of SEQ ID NO: 109-120.

In another aspect, the present disclosure provides a heavy chain variable region (V _H ) comprising (i) at least 90% identity with the amino acid sequence according to SEQ ID NO: 95; and (ii) at least 90% identity with the amino acid sequence according to SEQ ID NO: 96. provides an isolated antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure, comprising a light chain variable region (V _L ) comprising the identity of an antibody or antigen-binding fragment thereof; The sexual fragment binds to the antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus or hepatitis delta virus.

In further embodiments, (i) the VH comprises at least 95% identity with the amino acid sequence according to SEQ ID NO: 95, and/or (ii) the VL comprises at least 95% identity with the amino acid sequence according to SEQ ID NO: 96. In certain embodiments, the antibody or antigen-binding fragment comprises the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 according to SEQ ID NOs: 97-102, respectively.

In certain embodiments, the VH comprises or consists of the amino acid sequence according to SEQ ID NO:95 and the VL comprises or consists of the amino acid sequence according to SEQ ID NO:96.

Fc Portion In some embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of this disclosure include an Fc portion. In certain embodiments, the Fc portion may be derived from human origin, eg, from human IgG1, IgG2, IgG3, and/or IgG4. In certain embodiments, the antibody or antigen-binding fragment may include an Fc portion derived from human IgG1.

As used herein, the term "Fc portion" begins at the hinge region immediately upstream of the papain cleavage site (e.g., residue 216 of native IgG with the first residue 114 of the heavy chain constant region) and Refers to a sequence that includes or is derived from the portion of an immunoglobulin heavy chain that ends at the C-terminus of the globulin heavy chain. Thus, the Fc portion may be a complete Fc portion or a portion thereof (eg, a domain). In certain embodiments, the complete Fc portion includes a hinge domain, a CH2 domain, and a CH3 domain (eg, EU amino acids 216-446). An additional lysine residue (K) may be present at the extreme C-terminus of the Fc portion, but is often cleaved from the mature antibody.

Amino acid positions within the Fc portion herein are numbered according to the Kabat EU numbering system. See, eg, Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Dept. Health and Human Services, 1983 and 1987. Amino acid positions in the Fc portion can also be numbered according to the IMGT numbering system (including C domain unique numbering and exon numbering) and the Kabat numbering system.

In some embodiments, the Fc portion comprises at least one of a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a portion of a variant, or a fragment thereof. . In some embodiments, the Fc portion includes at least a hinge domain, a CH2 domain, or a CH3 domain. In further embodiments, the Fc portion is a complete Fc portion. The amino acid sequence of an exemplary Fc portion of the human IgG1 isotype is provided in SEQ ID NO: 137. The Fc portion may include one or more amino acid insertions, deletions, or substitutions relative to the naturally occurring Fc portion. For example, at least one or a portion of the hinge domain, CH2 domain, or CH3 domain may be deleted. For example, the Fc portion may be (i) a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (ii) a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). , (iii) a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iv) a hinge domain (or a portion thereof), (v) a CH2 domain (or a portion thereof), or ( vi) It may contain or consist of a CH3 domain or a part thereof.

The Fc portions of the present disclosure can be modified so that the portions have the amino acid sequence of a naturally occurring immunoglobulin molecule, while retaining or enhancing at least one desired function conferred by the naturally occurring Fc portion. It can be made to vary from the Fc part. Such functions include, for example, binding to Fc receptors (FcR), regulation of antibody half-life (e.g., by binding to FcRn), ADCC function, binding to protein A, binding to protein G, and complementation. Includes physical union. Some of the naturally occurring Fc moieties involved in such functions have been described in the art.

For example, to activate the complement cascade, a complex of C1q proteins can bind at least two IgG molecules or one IgM molecule when an immunoglobulin molecule binds to an antigenic target (Ward, E. S. , and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, D. R. described that the heavy chain region containing amino acid residues 318-337 is involved in complement fixation (Mol. Immunol. 22 (1985) 161-206). Duncan, A. R. and Winter, G. (Nature 332 (1988) 738-740) reported that by site-directed mutagenesis, Glu318, Lys320, and Lys322 form the binding site for C1q. The role of Glu318, Lys320, and Lys322 residues in C1q binding was confirmed by the ability of short synthetic peptides containing these residues to inhibit complement-mediated cytolysis.

For example, FcR binding can be mediated by the interaction of the Fc portion (of the antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on cells, including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and act through antibody-dependent cell-mediated cytotoxicity (ADCC; Van de Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). , has been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and various other cellular targets (e.g. tumor cells) coated with the corresponding antibodies. FcR is defined by its specificity for an immunoglobulin class; the Fc receptor for IgG antibodies is called FcγR, for IgE FcεR, for IgA FcαR, etc., and the neonatal Fc receptor is called FcRn. Ru. Fc receptor binding is described, for example, in Ravetch, J. V., and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., et al., Immunomethods 4 (1994) 25-34; de Haas , M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76 (1998) 231-248.

Cross-linking of receptors (FcγR) by the Fc domain of natural IgG antibodies has a wide range of effects, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as control of immune complex clearance and antibody production. induces effector functions. Fc moieties that provide cross-linking of receptors (eg, FcγR) are contemplated herein. Three classes of Fcγ have been characterized to date in humans: (i) FcγRI, which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils; (CD64), (ii) binds with moderate to low affinity to complexed IgG, is widely expressed, especially on leukocytes, and is thought to have a central role in antibody-mediated immunity, including FcγRIIA, FcγRIIB, and FcγRIIC. FcγRII (CD32), which can be divided into two groups and which exert different functions in the immune system but bind IgG-Fc with similar low affinity and the extracellular domains of these receptors are highly homologous; (iii) FcγRIIIA, which binds IgG with moderate to low affinity and is found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and is thought to mediate ADCC, and neutrophils; FcγRIII (CD16) was found in two forms of FcγRIIIB that are highly expressed in the human body.

FcγRIIA is found in many cells involved in killing (eg, macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcγRIIB appears to play a role in the inhibitory process and is found in B cells, macrophages, as well as mast cells and eosinophils. Importantly, it has been shown that 75% of all FcγRIIB is found in the liver (Ganesan, L. P. et al., 2012: "FcγRIIb on liver sinusoidal endothelium clears small immune complexes," Journal of Immunology 189: 4981 -4988). FcγRIIB is abundantly expressed in liver sinusoidal endothelium, called LSEC, and Kupffer cells of the liver, and LSEC is a major site of small immune complex clearance (Ganesan, L. P. et al., 2012: FcγRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988).

In some embodiments, the antibodies and antigen-binding fragments thereof disclosed herein include an Fc portion, particularly an Fc region, for binding to FcγRIIb, such as an IgG-type antibody. Furthermore, as described in Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933 , it is possible to enhance FcγRIIB binding by manipulating the Fc portion and introducing mutations S267E and L328F. Thereby, clearance of immune complexes can be enhanced (Chu, S., et al., 2014: Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor FcγRIIb. Am J Respir Crit, American Thoracic Society International Conference Abstracts). In some embodiments, the antibodies of the present disclosure or antigen-binding fragments thereof are particularly useful in Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc -engineered antibodies. Contains an engineered Fc portion with mutations S267E and L328F as described in Molecular Immunology 45, 3926-3933.

For B cells, FcγRIIB appears to function to suppress further immunoglobulin production and isotype switching, for example to the IgE class. For macrophages, FcγRIIB appears to inhibit phagocytosis as mediated by FcγRIIA. For eosinophils and mast cells, the b form may help suppress activation of these cells through binding of IgE to its isolated receptors.

With respect to FcγRI binding, modification of at least one of E233-G236, P238, D265, N297, A327, and P329 of native IgG reduces binding to FcγRI. Substitution of IgG2 residues at positions 233-236 to the corresponding positions of IgG1 and IgG4 reduced the binding of IgG1 and IgG4 to FcγRI by a factor of ¹⁰ and reduced the binding of human monocytes to antibody-sensitized red blood cells. (Armour, KL, et al. Eur. J. Immunol. 29 (1999) 2613-2624).

Regarding FcγRII binding, reduced binding to FcγRIIA is found for at least one IgG mutation, eg E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414.

The two allelic forms of human FcγRIIA are the "H131" variant, which binds IgG1 Fc with high affinity, and the "R131" variant, which binds IgG1 Fc with low affinity. See, eg, Bruhns et al., Blood 113:3716-3725 (2009).

Regarding FcγRIII binding, for example, for at least one mutation of E233 to G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376, Reduced binding to FcγRIIIA is found. Mapping of binding sites on human IgG1 for Fc receptors, mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, R. L., et al., J. Biol. Chem. 276 (2001). 6591-6604.

The two allelic forms of human FcγRIIIA are the "F158" variant, which binds IgG1 Fc with low affinity, and the "V158" variant, which binds IgG1 Fc with high affinity. See, eg, Bruhns et al., Blood 113:3716-3725 (2009).

With regard to binding to FcγRII, two regions of native IgG Fc are involved: (i) the lower hinge region of IgG Fc, specifically amino acid residues L, L, G, G (234-237, EU numbering); and (ii) ) Adjacent regions of the CH2 domain of IgG Fc, particularly the loops and strands of the upper CH2 domain adjacent to the lower hinge region, e.g. the region of P331, appear to be involved in the interaction of FcγRII with IgG (Wines, B.D. , et al., J. Immunol. 2000; 164: 5313 - 5318). Furthermore, FcγRI appears to bind to the same site on IgG Fc, whereas FcRn and protein A bind to a different site on IgG Fc, which appears to be the CH2-CH3 interface (Wines, B.D., et al., J. Immunol. 2000; 164: 5313 - 5318).

In some embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure have an Fc portion that binds to (i.e., one or more) Fcγ receptors, such as human FcγRIIa, The Fc portion includes a mutation that increases binding affinity for human FcγRIIIa, or both (as compared to a reference Fc portion or an antibody comprising an Fc portion without the mutation). See, eg, Delillo and Ravetch, Cell 161(5):1035-1045 (2015) and Ahmed et al., J. Struc. Biol. 194(1):78 (2016). The Fc mutations and methodology are incorporated herein by reference. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include G236A; S239D; A330L; and I332E; or combinations comprising these, e.g., S239D/I332E; G236A/S239D/I332E; G236A/A330L/I332E (also referred to herein as "GAALIE");

In certain embodiments, the Fc portion may include or consist of at least a portion of an Fc portion that is involved in binding to FcRn (eg, to human FcRn). In certain embodiments, the Fc portion improves the binding affinity to FcRn, and in some embodiments thereby increases the in vivo half-life of the molecule comprising the Fc portion (e.g., compared to an unmodified reference (compared to the Fc portion or antibody). In certain embodiments, the Fc portion comprises or is derived from an IgG Fc and the half-life increasing mutations are M428L, N434S, N434H, N434A, N434S, M252Y, S254T, T256E, T250Q, P257I, Q311I, D376V , T307A, and E380A (EU numbering). In certain embodiments, the half-life increasing mutation comprises M428L/N434S (also referred to herein as "MLNS"). In certain embodiments, the half-life increasing mutations include M252Y/S254T/T256E. In certain embodiments, the half-life increasing mutation comprises T250Q/M428L. In certain embodiments, the half-life increasing mutation comprises P257I/Q311I. In certain embodiments, the half-life increasing mutation comprises P257I/N434H. In certain embodiments, the half-life increasing mutation comprises D376V/N434H. In certain embodiments, the half-life increasing mutations include T307A/E380A/N434A.

In some embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion that includes the substitution mutation M428L/N434S. In some embodiments, the binding protein includes an Fc portion that includes substitution mutations G236A/A330L/I332E. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include the G236A mutation, the A330L mutation, and the I332L mutation (GAALIE), and include the S239D mutation. Contains no Fc portion. In some embodiments, the Fc portion includes Ser at position 239. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion that includes the substitution mutations M428L/N434S and G236A/A330L/I332E. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion that includes the substitution mutations M428L/N434S and G236A/S239D/A330L/I332E. . In certain further embodiments, the Fc portion does not include any substitution mutations except M428L/N434S and G236A/S239D/A330L/I332E.

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure are by any one of the exemplary anti-HBV antibodies disclosed herein. and/or CDRs and/or variable domains and/or heavy and/or light chains (antibodies HBC34, HBC34v7, HBC34v23, HBC34v31, HBC34v32, HBC34v33, HBC34v34, HBC34v35 [disclosed herein) in PCT Publication No. WO2017/060504. HBC antibody variants containing substitution mutations at position 40 of the light chain (e.g., substitution of natural cysteine with alanine, serine, etc.)], as well as G236A, A330L, and I332E (GAALIE) mutations. The Fc portion may further include the M428L/N434S (MLNS) mutation. In certain embodiments, the Fc portion does not include S239D.

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include the CDRH1 amino acid sequence according to SEQ ID NO: 34, the CDRH2 amino acid sequence according to SEQ ID NO: 35 or 66, CDRH3 amino acid sequence according to No. 36, CDRL1 amino acid sequence according to SEQ ID No. 37, CDRL2 amino acid sequence according to SEQ ID No. 38 or 39, and CDRL3 amino acid sequence according to SEQ ID No. 58 or 40, and an Fc portion containing the GAALIE mutation. In certain embodiments, the Fc portion further comprises an MLNS mutation.

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure have a heavy chain variable domain (VH) amino acid according to any one of SEQ ID NO: 41 or 67. and a light chain variable domain (VL) amino acid sequence according to any one of SEQ ID NOs: 42, 59, 65, 89, 90, and 111-120, and an Fc portion that includes a GAALIE mutation. In certain embodiments, the Fc portion further comprises an MLNS mutation.

In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises a heavy chain amino acid sequence according to SEQ ID NO: 138 or 91.

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include the CDRH1 amino acid sequence according to SEQ ID NO: 97, the CDRH2 amino acid sequence according to SEQ ID NO: 98, or the CDRH2 amino acid sequence according to SEQ ID NO: 99. CDRH3 amino acid sequence according to SEQ ID NO: 100, CDRL2 amino acid sequence according to SEQ ID NO: 100, and CDRL3 amino acid sequence according to SEQ ID NO: 102, and an Fc portion containing the GAALIE mutation. In certain embodiments, the Fc portion further comprises an MLNS mutation.

In any of the embodiments of the present disclosure, a binding protein of the present disclosure includes an Fc portion that includes a GAALIE mutation and is compared to a reference polypeptide (i.e., a polypeptide that may be a binding protein that includes an Fc portion that does not include a GAALIE mutation). has enhanced binding to human FcγRIIa and/or human FcγRIIIa.

In certain embodiments, the reference polypeptide comprises an Fc portion that is a wild-type Fc portion or comprises one or more substitution mutations (or insertions or deletions), provided that the substitution mutations Not GAALIE. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include HBC34v35 antibodies that include GAALIE and MLNS mutations, and the reference polypeptide is HBC34v35 ( (including a wild-type Fc portion of the same isotype as the Fc portion of an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure). In certain embodiments, the reference polypeptide does not contain substitution mutations known or believed to affect binding to human FcγRIIa and/or human FcγRIIIa.

Binding between polypeptides, such as binding between an Fc portion (or a binding protein comprising the same) and a human Fcγ receptor, such as human FcγRIIA, human FcγRIIIA, or human Fc FcγRIIB, or a complement protein such as C1q. , can be determined or detected by methods known in the art. For example, a biolayer interferometry (BLI) assay is performed using an Octet® RED96 (ForteBio, Fremont, California USA) instrument according to the manufacturer's instructions to detect a first polypeptide of interest, e.g. HBC34v35 containing a GAALIE mutation) and a second polypeptide of interest (e.g. FcγRIIA(H131), FcγRIIA(R131), FcγRIIIA(F158), FcγRIIIA(V158), or FcγRIIb) captured on a sensor substrate. Real-time association and dissociation can be determined.

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion that includes a GAALIE mutation and a reference molecule that includes an Fc portion that does not include a GAALIE mutation. has enhanced binding to human FcγRIIA (H131), human FcγRIIA (R131), human FcγRIIIA (F158), human FcγRIIIA (V158), or any combination thereof as compared to the polypeptide. In certain embodiments, enhanced binding is an increase in signal shift relative to a reference binding protein in a BLI assay (e.g., one or more of a higher peak signal, a greater association rate, a slower dissociation rate, or a greater area under the curve). determined by In certain embodiments, the BLI assay involves the use of an Octet® RED96 (ForteBio, Fremont, California USA) device. In a further embodiment, the BLI assay comprises a tagged human FcγR captured on an anti-pentatag sensor and exposed to binding protein. In some embodiments, the binding protein comprises an IgG Fab, and the BLI assay connects the captured human FcγR to an antibody or antigen-binding fragment in the presence of an anti-IgG Fab binding fragment that cross-links the binding protein through the Fab fragment. Further including exposing.

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion that includes a GAALIE mutation and has an enhanced Fc region as compared to a reference polypeptide. with binding to human FcγRIIA (H131), human FcγRIIA (R131), human FcγRIIIA (F158), and/or human FcγRIIIA (V158), the enhanced binding is associated with a signal shift observed using the reference antibody. Includes a signal shift (in nanometers) of a BLI assay that is 1.5x, 2x, 2.5x, 3x, or greater.

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion that includes a GAALIE mutation and has an enhanced Fc region as compared to a reference polypeptide. Has binding to human FcγRIIA (H131), human FcγRIIA (R131), human FcγRIIIA (F158), and human FcγRIIIA (V158).

In any of the embodiments of the present disclosure, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion that includes a GAALIE mutation and is compared to a reference polypeptide. Has reduced binding to human FcγRIIB. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion that includes a GAALIE mutation, e.g., a statistical increase relative to baseline in a BLI assay. FcγRIIB does not bind to human FcγRIIB, as determined by the absence of a statistically significant signal shift.

In any of the embodiments of the present disclosure, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion that includes a GAALIE mutation and is compared to a reference polypeptide. Has reduced binding to human C1q (complement protein). In certain embodiments, the binding protein comprises an Fc portion that includes a GAALIE mutation and does not bind human C1q as determined by the absence of a statistically significant signal shift relative to baseline in a BLI assay.

In any of the embodiments of the present disclosure, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion that includes a GAALIE mutation and contains a reference polypeptide (i.e., GAALIE The polypeptide, which may be an antibody or an antigen-binding fragment thereof comprising an Fc portion that does not contain the mutation, activates human FcγRIIA, human FcγRIIIA, or both to a greater extent. In certain embodiments, the reference polypeptide comprises a wild-type Fc portion or an Fc portion that includes one or more substitution mutations, provided that the substitution mutations are not GAALIE. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HBC34v35 antibody with a GAALIE mutation (and other substitution mutations, which may be, for example, MLNS), and the reference polypeptide comprises an HBC34v35 antibody with a wild-type Fc portion. It is.

Activation of human FcγRs can be determined or detected using methods known in the art. For example, a well-validated commercially available bioreporter assay allows the binding of HBsAg-specific binding proteins to Jurkat effector cells stably expressing (i) the FcγR of interest and (ii) the firefly luciferase reporter under the control of NFAT response elements. (Promega, Cat. no: G9798) with recombinant HBsAg (Engelix B, GlaxoSmithKline). Fc binding to FcγR expressed on the cell surface drives NFAT-mediated luciferase reporter gene expression. Luminescence is then measured by a luminometer (eg, Bio-Tek) using the Bio-Glo-™ Luciferase Assay Reagent (Promega) according to the manufacturer's instructions. Activation is expressed as the average luminescence units (RLU) relative to background by applying the following formula: [RLU at concentration [x] of binding protein (e.g. mAb) - RLU of background]. Ru.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises an Fc portion that includes a GAALIE mutation and comprises human FcγRIIA (H131), human FcγRIIIA (F158), and/or human FcγRIIIA ( V158). In certain embodiments, a greater degree of activation refers to higher peak luminescence and/or greater area under the luminescence curve as determined using the luminescent bioreporter assays described herein. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an Fc portion that includes a GAALIE mutation and has human FcγRIIA (H131), human FcγRIIA (R131), and human FcγRIIIA (F158) to a greater extent than the reference polypeptide. , and a greater degree of activation may be represented by a peak RLU of 1.5-fold, 2-fold, 2.5-fold, 3-fold, or greater than the peak RLU observed using the reference polypeptide. can.

In any of the embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises an Fc portion that includes a GAALIE mutation, and the absence of a statistically significant and/or measurable RLU in the luminescent bioreporter assay described above. does not activate human FcγRIIB as determined by .

In any of the embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises an Fc portion that includes a GAALIE mutation and activates human natural killer (NK) cells to a greater extent than the reference polypeptide in the presence of HBsAg. do. In certain embodiments, NK cell activation is determined by CD107a expression (eg, by flow cytometry). In certain embodiments, the NK cell comprises a cell comprising a FcγRIIIa genotype that is V158/V158 homozygous, F158/F158 homozygous, or V158/F158 heterozygous.

Any antibody or antigen-binding fragment thereof that includes an Fc portion that includes a GAALIE mutation according to the present disclosure has the characteristics described herein, such as enhanced human FcγRIIA and/or human FcγRIIIA as compared to a reference polypeptide. binding, reduced binding to human FcγRIIB (and/or lack of binding to human FcγRIIB) compared to a reference polypeptide, reduced binding to human C1q (and/or activating FcγRIIA, human FcγRIIIA, or both to a greater extent than the reference polypeptide; not activating human FcγRIIB; and/or the reference polypeptide in the presence of HBsAg. activating human natural killer (NK) cells to a greater extent (e.g., an antibody containing an Fc portion specific for HBsAg and without a GAALIE mutation). Recognizes what can be done.

Alternatively or additionally, the Fc portion of the antibodies of the present disclosure or antigen-binding fragment thereof may include at least a portion known in the art necessary for protein A binding, and/or the Fc portion of the antibodies of the present disclosure may include , comprising at least a portion of an Fc molecule known in the art required for protein G binding. In some embodiments, the retained function includes exclusion of HBsAg and HBVg. Thus, in certain embodiments, the Fc portion includes at least the portion known in the art necessary for FcγR binding. Thus, as outlined above, the Fc portion includes (i) the lower hinge region of the native IgG Fc, specifically amino acid residues L, L, G, G (234-237, EU numbering), and (ii ) the adjacent regions of the CH2 domain of the native IgG Fc, in particular the loops and strands of the upper CH2 domain adjacent to the lower hinge region, e.g. the region of P331, e.g. at least 3,4 of the upper CH2 domain of the native IgG Fc around P331; , 5, 6, 7, 8, 9, or 10 contiguous amino acids, such as between amino acids 320-340 (EU numbering) of native IgG Fc.

In some embodiments, antibodies or antigen-binding fragments thereof according to the present disclosure include an Fc region. As used herein, the term "Fc region" refers to the portion of an immunoglobulin formed by two or more Fc portions of an antibody heavy chain. For example, the Fc region can be a monomeric or "single chain" Fc region (ie, a scFc region). A single-chain Fc region consists of Fc portions linked into a single polypeptide chain (eg, encoded by a single contiguous nucleic acid sequence). Exemplary scFc regions are described in WO2008/143954A2, incorporated herein by reference. The Fc region may be or include a dimeric Fc region. "Dimeric Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two separate immunoglobulin heavy chains. A dimeric Fc region can be a homodimer of two identical Fc regions (e.g., naturally occurring immunoglobulin Fc regions) or a heterodimer of two non-identical Fc regions (e.g., one Fc monomer of a dimeric Fc region is attached to another Fc region). The monomer may contain at least one amino acid modification (e.g., a substitution, deletion, insertion, or chemical modification) that is not present, or one Fc monomer may be truncated relative to the other).

Certain embodiments include antibodies and antigen-binding fragments having a heavy chain according to SEQ ID NO: 91 or SEQ ID NO: 92 (e.g., VH-hinge-CH1-CH2-CH3) and a light chain (such as VH-hinge-CH1-CH2-CH3) according to SEQ ID NO: 93 or SEQ ID NO: 94. ie, VL-CL) and antigen-binding fragments. In certain embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:91 and a light chain according to SEQ ID NO:93. In other embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:92 and a light chain according to SEQ ID NO:94. In other embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:91 and a light chain according to SEQ ID NO:94. In other embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:92 and a light chain according to SEQ ID NO:93. In some embodiments, the antibody or antigen-binding fragment comprises or consists of a heavy chain according to SEQ ID NO: 129. In some embodiments, the antibody or antigen-binding fragment comprises or consists of a heavy chain according to SEQ ID NO: 138. These sequences are provided in the sequence listing.

Fc portions of the present disclosure may include Fc sequences or regions of the same or different classes and/or subclasses. For example, the Fc portion can be derived from immunoglobulins of the IgG1, IgG2, IgG3, or IgG4 subclasses (eg, human immunoglobulins), or from any combination thereof. In certain embodiments, the Fc portions of the Fc region are of the same class or subclass. However, the Fc region (or one or more Fc portions of an Fc region) may be chimeric, whereby a chimeric Fc region may include Fc portions derived from different immunoglobulin classes and/or subclasses. For example, at least two of the Fc portions of a dimeric or single chain Fc region may be from different immunoglobulin classes and/or subclasses. In certain embodiments, a dimeric Fc region may include sequences from two or more different isotypes or subclasses, such as SEED bodies ("strand-exchange engineered domains"). See Davis et al., Protein Eng. Des. Sel. 23(4):195 (2010).

Additionally or alternatively, a chimeric Fc region may have one or more chimeric Fc portions. For example, a chimeric Fc region or portion may include one or more portions derived from immunoglobulins of a first subclass (e.g., IgG1, IgG2, or IgG3 subclass), while the remainder of the Fc region or portion is of a different subclass. It is something. For example, the Fc region or portion of an Fc polypeptide may include a CH2 and/or CH3 domain derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG4 subclass) and a CH2 and/or CH3 domain derived from an immunoglobulin of a second subclass (e.g., IgG3 subclass). It may contain a hinge region derived from an immunoglobulin. For example, the Fc region or portion may include a hinge and/or CH2 domain derived from an immunoglobulin of a first subclass (e.g., IgG4 subclass) and an immunoglobulin of a second subclass (e.g., IgG1, IgG2, or IgG3 subclass). May contain a CH3 domain. For example, a chimeric Fc region includes an Fc portion (e.g., a complete Fc portion) from an immunoglobulin of a first subclass (e.g., the IgG4 subclass) and an Fc portion (e.g., a complete Fc portion) from an immunoglobulin of a second subclass (e.g., the IgG1, IgG2, or IgG3 subclass). It may include an Fc portion. For example, the Fc region or portion can include a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, the Fc region or portion can include a CH1 domain and a CH2 domain from an IgG4 molecule, and a CH3 domain from an IgG1 molecule. For example, the Fc region or portion can include a portion of the CH2 domain from a particular subclass of antibodies, eg, CH2 domain EU positions 292-340. For example, the Fc region or portion can include amino acids 292-340 of CH2 derived from an IgG4 portion and the remainder of CH2 derived from an IgG1 portion (alternatively, CH2 292-340 is derived from an IgG1 portion and CH2 the remainder may be derived from the IgG4 portion).

It is also recognized that any antibody, antigen binding portion, or Fc region or portion of this disclosure may be of any allotype and/or haplotype. For example, human immunoglobulin G allotypes include those disclosed in Jefferis and LeFranc, mAbs 1(4):1-7 (2009); G2m[23(n)]; G3m[21(g1); 28(g5); 11(b0); 5(b2); 13(b3); 14(b4 ); 10(b5); 15(s); 16(t); 6(c3); 24(c5); 26(u); and 27(v)]; A2m(1 and 2); and Km(1 ; 2; and 3) and haplotypes, and resulting amino acid sequences, and combinations thereof are incorporated herein by reference. In certain embodiments, antibodies, antigen-binding fragments, or Fc regions or The portion contains the IgG1 allotype g1m17,k1.

Additionally, the Fc region or portion (also or alternatively) includes, for example, a chimeric hinge region. For example, a chimeric hinge is, for example, partially derived from IgG1, IgG2, or IgG4 molecules (eg, upper and lower middle hinge sequences) and partially derived from IgG3 molecules (eg, middle hinge sequences). In another example, the Fc region or portion can include a chimeric hinge derived partially from IgG1 molecules and partially from IgG4 molecules. In another example, a chimeric hinge can include upper and lower hinge domains from an IgG4 molecule and a middle hinge domain from an IgG1 molecule. Such a chimeric hinge can be created, for example, by introducing a proline substitution (Ser228Pro) at position EU228 of the middle hinge domain of the IgG4 hinge region. In another embodiment, the chimeric hinge may include amino acids at positions EU233-236 from an IgG2 antibody and/or the Ser228Pro mutation, with the remaining amino acids of the hinge being from an IgG4 antibody (eg, a chimeric hinge of the sequence ESKYGPPCPPCPAPPVAGP). Additional chimeric hinges that can be used in the Fc portion of antibodies according to the present disclosure are described in US2005/0163783A1.

In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein, the Fc portion or region is an amino acid derived from a human immunoglobulin sequence (e.g., from the Fc region or portion of a human IgG molecule). Contains or consists of an array. However, the polypeptide may include one or more amino acids from another mammalian species. For example, a primate Fc portion or a primate binding site may be included in the subject polypeptide. Alternatively, one or more murine amino acids may be present in the Fc portion or region.

Nucleic Acid Molecules In another aspect, the present disclosure provides nucleic acid molecules comprising polynucleotides encoding antibodies or antigen-binding fragments thereof according to the present disclosure.

Table 4 shows nucleotide sequences encoding exemplary V _H , V _L , CH, CL, HC, and LC according to the present disclosure.

Due to the redundancy of the genetic code, this disclosure also includes sequence variants of these nucleic acid sequences, especially such sequence variants that encode the same amino acid sequence.

In certain embodiments, the polynucleotide or nucleic acid molecule comprises a nucleotide sequence that shares at least 80% identity with a nucleotide sequence according to any one of SEQ ID NOs: 103-110 and 130-136, and the nucleotide sequence is Codon optimized for expression by host cells.

In certain embodiments, a nucleic acid molecule according to the present disclosure comprises or consists of a nucleic acid sequence according to any one of SEQ ID NOs: 103-110 and 130-136.

In certain embodiments, the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 103 encoding a V _H and a nucleotide sequence according to SEQ ID NO: 105 encoding a V _L. In other embodiments, the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 103 encoding a V _H and a nucleotide sequence according to SEQ ID NO: 104 encoding a V _L. In other embodiments, the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 108 encoding a V _H and a nucleotide sequence according to SEQ ID NO: 109 encoding a V _L.

Also provided herein is a polynucleotide encoding an antibody or antigen-binding fragment comprising or consisting of the nucleotide sequence according to _SEQ ID NO: 103 encoding a _VH and the nucleotide sequence according to SEQ ID NO: 110 encoding a VL, The antibody or antigen-binding fragment thus obtained binds to the antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus or hepatitis delta virus.

In any of the embodiments of the present disclosure, the polynucleotide may comprise the nucleotide sequence according to SEQ ID NO: 130 encoding CH1-hinge-CH2-CH3 and/or HC(VH-CH1-hinge-CH3-CH3). It contains the nucleotide sequence encoding SEQ ID NO: 131. In some embodiments, the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 132 encoding CL and/or a nucleotide sequence according to SEQ ID NO: 133 encoding LC (VL-CL). In other embodiments, the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 134 encoding CL and/or a nucleotide sequence according to SEQ ID NO: 135 or SEQ ID NO: 136 encoding LC (VL-CL).

Vectors Further included within the scope of this disclosure are vectors, such as expression vectors, that include nucleic acid molecules according to this disclosure.

The term "vector" refers to a construct that includes a nucleic acid molecule. Vectors in the context of this disclosure are suitable for incorporating or containing desired nucleic acid sequences. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A storage vector is a vector that allows convenient storage of nucleic acid molecules. That is, the vector may contain sequences corresponding to a desired antibody or antibody fragment thereof according to the present invention.

As used herein, "expression vector" refers to a DNA construct that includes a nucleic acid molecule operably linked to suitable control sequences capable of effecting expression of the nucleic acid molecule in a suitable host. Such control sequences include promoters that effect transcription (e.g., heterologous promoters), appropriate operator sequences that control such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that control termination of transcription and translation. Contains arrays. Any of the elements of the expression vector that contribute to transcription of the nucleic acid molecule of interest may be heterologous to the vector. The vector may be a plasmid, a phage particle, a virus, or simply a possible genomic insert. Once transformed into a suitable host, the vector replicates and functions independently of the host genome, or in some cases integrates into the genome itself. As used herein, "plasmid," "expression plasmid," "virus," and "vector" are often used interchangeably.

A cloning vector is a vector that typically contains cloning sites and is used to incorporate nucleic acid sequences into the vector. The cloning vector may be, for example, a plasmid vector or a bacteriophage vector.

The transfer vector may be a vector suitable for transferring a nucleic acid molecule into a cell or an organism, such as a viral vector. A vector in the context of this disclosure may be, for example, an RNA vector or a DNA vector. A vector may be a DNA molecule. For example, a vector within the meaning of this application contains sequences suitable for the replication of the vector, such as a cloning site, a selection marker such as an antibiotic resistance factor, and an origin of replication. In some embodiments, vectors in the context of this application are plasmid vectors. In certain such embodiments, the vector comprises a lentiviral vector or a retroviral vector.

Cells In a further aspect, the present disclosure also provides cells (also referred to as "host cells") that express antibodies, antigen-binding fragments, or fusion proteins according to the present disclosure, or that include vectors or polynucleotides according to the present disclosure.

Examples of such cells include, but are not limited to, eukaryotic cells such as yeast cells, animal cells, insect cells, plant cells, and prokaryotic cells, including E. coli. In some embodiments, the cell is a mammalian cell. In certain such embodiments, the cells are mammalian cell lines, such as CHO cells (e.g., DHFR-CHO cells (Urlaub et al., PNAS 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells). , PER.C6 cells, Y0 cells, Sp2/0 cells, NS0 cells, human hepatocytes such as Hepa RG cells, myeloma cells, or hybridoma cells. Other examples of mammalian host cell lines include mouse Sertoli cells. cells (e.g. TM4 cells), SV40-transformed monkey kidney CV1 line (COS-7), baby hamster kidney cells (BHK), African green monkey kidney cells (VERO-76), monkey kidney cells (CV1), human cervix Cancer cells (HELA), human lung cells (W138), human hepatocytes (Hep G2), dog kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), mouse mammary tumor (MMT 060562), TRI cells, MRC5 cells , and FS4 cells. Suitable mammalian host cell lines for the production of antibodies include, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), Also includes those described on pp. 255-268 (2003).

In certain embodiments, the host cell is a prokaryotic cell, such as E. coli. Expression of peptides in prokaryotic cells such as E. coli is well established (see, eg, Pluckthun, A. Bio/Technology 9:545-551 (1991)). For example, antibodies are produced in bacteria, especially when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, eg, US Patent Nos. 5,648,237, 5,789,199, and 5,840,523.

Useful insect cells expressing antibodies of the present disclosure or antigen-binding fragments thereof are known in the art, such as Spodoptera frugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells. , and Spodoptera frugipera SfSWT01 "Mimic™" cells. See, eg, Palmberger et al., J. Biotechnol. 153(3-4):160-166 (2011). A number of baculovirus strains have been identified that are used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

Eukaryotic microorganisms, such as filamentous fungi or yeast, are also suitable hosts for cloning or expressing protein-encoding vectors and have "humanized" glycosylation pathways, allowing for partial or complete human glycosylation. Included are strains of fungi and yeast that result in the production of patterned antibodies. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006).

Plant cells can also be utilized as hosts for expressing the disclosed antibodies or antigen-binding fragments thereof. For example, PLANTIBODIES™ technology (e.g., U.S. Patent Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; ) employs transgenic plants to produce antibodies.

Any protein expression system compatible with this disclosure may be used to produce the disclosed antibodies or antigen-binding fragments thereof. Suitable expression systems include transgenic animals as described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

In certain embodiments, cells can be transfected with vectors herein, including expression vectors. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (eg, mRNA) molecule, into a cell, eg, a eukaryotic cell. In the context of this specification, the term "transfection" encompasses any method known to those skilled in the art for the introduction of nucleic acid molecules into cells, such as eukaryotic cells, including mammalian cells. Such methods include, for example, electroporation, lipofection based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymers such as DEAE dextran or polyethyleneimine. transfection based on . In certain embodiments, the introduction is non-viral.

Furthermore, cells of the present disclosure can be stably or transiently transfected with vectors according to the present disclosure, eg, to express antibodies according to the present disclosure or antigen-binding fragments thereof. In such embodiments, the cells are stably transfected with a vector described herein encoding a binding protein. Alternatively, cells can be transiently transfected with vectors according to the present disclosure encoding binding proteins according to the present specification. In any of the embodiments of this disclosure, the polynucleotide may be heterologous to the host cell.

In a related aspect, the disclosure provides a method of producing an antibody or antigen-binding fragment thereof, the method comprising: producing an antibody or antigen-binding fragment thereof under conditions and for a period of time sufficient to produce the antibody or antigen-binding fragment thereof. including culturing host cells.

Accordingly, the present disclosure also provides recombinant host cells that heterologously express the disclosed antibodies or antigen-binding fragments thereof. For example, the cell may be of a different species than the one from which the antibody was wholly or partially obtained (eg, a CHO cell expressing a human antibody or an engineered human antibody). In some embodiments, the host cell cell type does not naturally express the antibody or antigen-binding fragment. Additionally, the host cell may not exist in the native state of the antibody or antigen-binding fragment (or in the native state of the parent antibody from which the antibody or antigen-binding fragment is engineered or derived). Post-translational modifications (PTMs, such as glycosylation or fucosylation) may be imparted. Such PTMs may result in functional differences (eg, reduced immunogenicity). Thus, an antibody or antigen-binding fragment of the present disclosure produced by a host cell disclosed herein may have one or more post-translational modifications that distinguish it from the antibody in its native state (or parent antibody). (e.g., a human antibody produced by a CHO cell may contain more than (may include post-translational modifications).

Optional Additional Characteristics of Antibodies or Antigen-Binding Fragments Antibodies and antigen-binding fragments of the present disclosure can be coupled to drugs, for example, for delivery to a treatment site, or to facilitate imaging of a site containing cells of interest. may be linked to a detectable label. Methods of linking antibodies to drugs and detectable labels are known in the art, as are methods of imaging using detectable labels. Labeled antibodies can be employed in a wide variety of assays employing a wide variety of labels. Detection of the formation of an antibody-antigen complex between an antibody (or antigen-binding fragment or fusion protein) of the present disclosure and an epitope of interest on HBsAg, particularly in the antigenic loop region of HBsAg, can detect the formation of an antibody-antigen complex in the antigenic loop region of HBsAg. This can be facilitated by combining the Suitable detection means include radionuclides, enzymes, coenzymes, fluorescent agents, chemiluminescent agents, dyes, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and other labels. Includes the use of Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include 125I, 131I, 35S, or 3H. Such labeled agents can be used in a variety of known assays, such as radioimmunoassays, enzyme immunoassays, such as ELISA, fluorescent immunoassays, and others. Accordingly, labeled antibodies and antigen-binding fragments according to the present disclosure are described, for example, in U.S. Pat. It can be used in the assays described in No.

An antibody or antigen-binding fragment thereof according to the present disclosure may be conjugated to a therapeutic moiety, such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or isotope. Examples of radioactive isotopes include, but are not limited to, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi -213, Pd-109, Tc-99, In-111, etc. Such antibody conjugates can be used to modify a given biological response. Drug moieties should not be construed as limited to classical chemical therapeutic agents. For example, a drug moiety may be a protein or polypeptide that possesses the desired biological activity. Such proteins may include toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moieties to antibodies are known. For example, Arnon et al. (1985) "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy," in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp. 243-256; ed. Hellstrom et al. (1987) "Antibodies for Drug Delivery," in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pp. 623-653; Thorpe (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al. pp. 475-506 (Editrice Kurtis, Milano, Italy, 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, " in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al. (1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody or antigen-binding fragment thereof is conjugated to a second antibody or antibody fragment thereof (or a second fusion protein), as described in U.S. Pat. No. 4,676,980, and a heteroconjugate A gate can be formed. Additionally, a linker may be used between the label and the antibodies herein, as described, for example, in US Pat. No. 4,831,175. Antibodies, antigen-binding fragments, and fusion proteins may be synthesized using radioactive iodine, indium, yttrium, or other radioactive particles known in the art, as described, for example, in U.S. Pat. No. 5,595,721. It may also be directly labeled by The treatment may consist of a combination of treatment with conjugated or unconjugated antibodies and/or antigen-binding fragments administered simultaneously or subsequently, as described for example in WO 00/52031, WO 00/52473. It's okay.

The antibodies and antigen-binding fragments described herein may be attached to a solid support. Additionally, an antibody or functional antibody fragment thereof of the present disclosure can be chemically modified, eg, by covalent conjugation to a polymer, to increase its circulating half-life. Examples of polymers and methods of attaching them to peptides are shown in U.S. Patent Nos. 4,766,106; 4,179,337; 4,495,285; ing. In some embodiments, the polymer may be selected from polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula R(O-CH ₂ -CH ₂ ) _n O-R, where R may be hydrogen or a protecting group such as an alkyl or alkanol group. In certain embodiments, protecting groups can have 1-8 carbons. For example, the protecting group may be methyl. The symbol n is a positive integer. In one embodiment, n is 1-1,000. In another embodiment, n is 2-500. In some embodiments, the PEG has an average molecular weight selected from 1,000-40,000, 2,000-20,000, and 3,000-12,000. Furthermore, PEG may have at least one hydroxy group, for example PEG may have a terminal hydroxy group. For example, this is a terminal hydroxy group that is activated and reacts with a free amino group on the inhibitor. However, it is understood that the type and amount of reactive groups can be varied to achieve the covalently conjugated PEG/antibodies herein.

Water-soluble polyoxyethylated polyols may also be utilized in the context of the antibodies and antigen-binding fragments described herein. These include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and others. In one embodiment, POG is used. Without wishing to be bound by any theory, the glycerol backbone of polyoxyethylated glycerol is the same backbone as naturally occurring mono-, di-, and tri-glycerides, for example in animals and humans; are not necessarily considered foreign substances in living organisms. POG can have a molecular weight in the same range as PEG. Another drug delivery system that can be used to increase circulating half-life is liposomes. Methods of preparing liposome delivery systems are known to those skilled in the art. Other drug delivery systems are known in the art and are described, for example, in Poznansky et al. (1980) and Poznansky (1984), cited above.

Typically, the antibody or antigen-binding fragment will be present in a composition substantially free of other polypeptides, such as less than 90% (by weight), usually less than 60%, more usually less than 60% of the composition. is composed of less than 50% of other polypeptides.

Antibodies or antigen-binding fragments of the present disclosure may be immunogenic in non-human (or xenogeneic) hosts, such as mice. In particular, an antibody, antigen-binding fragment, or fusion protein may have an idiotope that is immunogenic in a non-human host, but not in a human host. In particular, such molecules of the present disclosure for human use cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, and are generally modified by humanization or by humanization. Contains molecules that cannot also be obtained from xenogeneic mice.

Production of Antibodies, Antigen-Binding Fragments, and Fusion Proteins Antibodies and antigen-binding fragments according to the present disclosure can be made by any method known in the art. For example, general methodologies for making monoclonal antibodies using hybridoma technology are known (Kohler, G. and Milstein, C., 1975; Kozbar et al. 1983). In one embodiment, an alternative EBV immortalization method described in WO2004/076677 is used.

In one embodiment, antibodies are produced using the methods described in WO2004/076677. In such methods, antibody-producing B cells are transformed with EBV and a polyclonal B cell activator. Additional stimulators of cell proliferation and differentiation may optionally be added during the transformation step to further enhance efficiency. These stimulants may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, but its use is not required. Immortalized B cells produced using these methods can be cultured and antibodies isolated therefrom using methods known in the art.

Another method for producing antibodies is described in WO2010/046775. In such methods, plasma cells are cultured in limited numbers or as single plasma cells in microwell culture plates. Antibodies can be isolated from plasma cell cultures. Additionally, RNA can be extracted from plasma cell cultures and PCR performed using methods known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR (reverse transcriptase PCR), sequenced, and cloned into an expression vector, which can then be transfected into HEK293T cells or other host cells. Cloning of nucleic acids in expression vectors, transfection of host cells, culturing of transfected host cells, and isolation of produced antibodies can be performed using any method known to those skilled in the art.

Antibodies may be further purified if necessary using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography. Methods for the purification of antibodies, eg, monoclonal antibodies, including techniques for producing pharmaceutical grade antibodies, are known in the art.

Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibodies, antibody fragments, or fusion proteins herein. The desired DNA sequence may be completely or partially synthesized using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

Any suitable host cell/vector system may be used for expression of DNA sequences encoding the disclosed antibody or fusion protein molecules or fragments thereof. Use of bacteria, e.g. E. coli and other microbial systems, in part for the expression of antibody fragments such as Fab and F(ab')2 fragments, and in particular Fv fragments and single chain antibody fragments, e.g. single chain Fv. You may do so. For production of larger antibody molecules, including complete antibody molecules, eukaryotic, eg, mammalian, host cell expression systems may be used. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER. Includes C6, NSO, myeloma, or hybridoma cells.

The present disclosure provides methods for culturing host cells containing vectors encoding nucleic acids of the present disclosure under conditions suitable for expression of proteins from DNA encoding the antibody molecules herein, and isolating the antibody molecules. Also provided are processes for the production of antibodies or antigen-binding fragments according to the present disclosure, including.

The antibody molecule or antibody fragment may contain only a heavy or light chain polypeptide, in which case only the sequences encoding the heavy or light chain polypeptide are used to transfect the host cell. There is a need. To produce a product containing both heavy and light chains, the cell line is transduced with two vectors, a first vector encoding the light chain polypeptide and a second vector encoding the heavy chain polypeptide. can be affected. Alternatively, a single vector containing sequences encoding light and heavy chain polypeptides is used.

Alternatively, antibodies and antigen-binding fragments according to the present disclosure can be obtained by (i) expressing a nucleic acid sequence according to the present disclosure in a host cell, e.g., by use of a vector according to the present disclosure, and (ii) producing the expressed desired product. may be produced by isolating. Additionally, the method may include (iii) purifying the isolated antibody or antigen-binding fragment. Transformed B cells and cultured plasma cells may be screened for cells that produce antibodies and antigen-binding fragments with the desired specificity or function.

Screening can be performed by any immunoassay, such as by ELISA, by staining of tissues or cells (including transfected cells), by neutralization assays, or by any number known in the art to identify the desired specificity or function. This may be done by one of the other methods. Assays may be selected based on simple recognition of one or more antigens, or may be selected further based on desired function, such as simply selecting neutralizing antibodies over antigen-binding antibodies; Characteristics of the target cells, such as their signaling cascades, their shape, their rate of proliferation, their ability to influence other cells, their response to the effects of other cells or other agents or changes in conditions, their differentiation Antibodies can be selected that can alter the state of, among other things.

Individual transformed B cell clones can then be produced from cultures of positive transformed B cells. Cloning steps to isolate individual clones from a mixture of positive cells may be performed using limiting dilution, micromanipulation, single cell deposition by cell sorting, or other methods known in the art.

Nucleic acids from cultured plasma cells can be isolated, cloned, and expressed in HEK293T cells or other known host cells using methods known in the art.

The immortalized B cell clones or transfected host cells described herein can be used to produce nucleic acids (DNA or mRNA) encoding monoclonal antibodies of interest for research in a variety of ways, e.g., as a source of monoclonal antibodies. It can be used as a source.

Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising an antibody that neutralizes hepatitis B virus and a pharmaceutically acceptable aqueous vehicle. It is understood that a vehicle is typically a suitable material for the storage, transport, formulation, and/or administration of pharmaceutically active compounds, particularly compounds such as antibodies according to the present disclosure. For example, the vehicle may be a physiologically acceptable liquid suitable for storage, transport, and/or administration of pharmaceutically active compounds, particularly antibodies according to the present disclosure.

The pharmaceutical compositions described herein are prepared for injection or infusion into a patient. In some embodiments, the compositions may be prepared for intravenous ("IV" or "i.v."), intraarterial, or intraventricular injection. In other embodiments, the composition may be prepared for intravenous, intraarterial, intraventricular, intramedullary, intraperitoneal, intrathecal, intraventricular injection. In certain embodiments, the compositions are prepared for subcutaneous ("SC" or "s.c.") injection. In certain embodiments, the compositions described herein are pharmaceutically acceptable sterile aqueous solutions that exhibit suitable pH, isotonicity, and stability for administration to human subjects. Suitable aqueous vehicles for formulating the compositions described herein include water (e.g., sterile water, USP Water for Injection), as well as sodium chloride for injection, Ringer's Injection, Lactated Ringer's Injection, and the like. Includes tension medium.

The pharmaceutical composition according to the present invention comprises an antibody selected from the HBV neutralizing antibodies according to the present invention. For example, in some embodiments, a pharmaceutical composition according to the present invention comprises (i) a heavy chain variable region (VH) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO: 41; (isolated) antibodies comprising a light chain variable region (VL) comprising at least 90% identity with an amino acid sequence according to any one of the following: 59, 89, or 90; The amino acid at position 40 is not cysteine, and the antibody or antigen-binding fragment thereof binds to the antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus and hepatitis delta virus.

In certain embodiments, (i) the VH comprises at least 95% identity with the amino acid sequence according to SEQ ID NO: 41, and/or (ii) the VL according to any one of SEQ ID NO: 59, 89, or 90. Contains at least 95% identity with the amino acid sequence.

In certain embodiments, the amino acid at position 40 of the VL is alanine. In certain embodiments, the amino acid at position 40 of the VL is serine. In certain embodiments, the amino acid at position 40 of VL is glycine.

In certain embodiments, the antibodies are SEQ ID NOs: (i) 34-36, 37, 38, and 40, respectively; (ii) 34, 66, 36, 37, 38, and 40, respectively; (iii) 34-34, respectively; 36, 37, 39, and 40; (iv) 34, 66, 36, 37, 39, and 40, respectively; (v) 34-36, 37, 38, and 58, respectively; (vi) 34, 66, 36, respectively. , 37, 38, and 58; (vii) 34-36, 37, 39, and 58, respectively; or (viii) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 by 34, 66, 36, 37, 39, and 58, respectively. , and CDRL3 sequences.

In certain embodiments, the VL comprises or consists of the amino acid sequence according to SEQ ID NO:89.

In certain embodiments, the VL comprises or consists of the amino acid sequence according to SEQ ID NO:90.

In certain embodiments, the VH comprises or consists of the amino acid sequence according to SEQ ID NO:41.

In certain embodiments, the VH comprises or consists of the amino acid sequence according to SEQ ID NO: 41 and the VL comprises or consists of the amino acid sequence according to SEQ ID NO: 89.

In certain embodiments, the VH comprises or consists of the amino acid sequence according to SEQ ID NO: 41 and the VL comprises or consists of the amino acid sequence according to SEQ ID NO: 90.

In certain embodiments, the antibodies include human antibodies and/or monoclonal antibodies.

In certain embodiments, the antibody is a multispecific antibody. In certain embodiments, the antibody is a bispecific antibody.

In certain embodiments, the antibody includes an Fc portion.

In certain embodiments, the Fc portion includes a mutation that enhances binding to (eg, human) FcRn as compared to a reference Fc portion that does not include the mutation.

In certain embodiments, the Fc portion includes a mutation that enhances binding to a (eg, human) FcγR (eg, FcγRIIa, FcγRIIIa, or both) as compared to a reference Fc portion that does not include the mutation.

In certain embodiments, the Fc portion is or is derived from an IgG isotype.

In certain embodiments, the mutation that enhances binding to FcRn is M428L, N434S, N434H, N434A, N434S, M252Y, S254T, T256E, T250Q, P257I, Q311I, D376V, T307A, E380A, or any of the following. Including combinations.

In certain embodiments, the mutations that enhance binding to FcRn are (i) M428L/N434S, (ii) M252Y/S254T/T256E, (iii) T250Q/M428L, (iv) P257I/Q311I, (v) P257I/N434H, (vi) D376V/N434H, (vii) T307A/E380A/N434A, or (viii) any combination of (i) to (vii).

In certain embodiments, mutations that enhance binding to FcRn include M428L/N434S.

In certain embodiments, mutations that enhance binding to FcγR include S239D, I332E, A330L, G236A, or any combination thereof.

In certain embodiments, the mutation that enhances binding to FcγR is (i) S239D/I332E, (ii) S239D/A330L/I332E, (iii) G236A/S239D/I332E, or (iv) G236A/A330L/ Contains I332E.

In certain embodiments, the mutation that enhances binding to FcγR comprises or consists of G236A/A330L/I332E. In some embodiments, the mutation that enhances binding to FcγR does not include S239D. In some embodiments, the Fc portion includes a natural Ser (S) at position 239.

In certain embodiments, the Fc portion includes amino acid substitution mutations: M428L, N434S, G236A, A330L, and I332E. In certain further embodiments, the Fc portion does not include additional mutations.

In certain embodiments, the antibody comprises a heavy chain (HC) amino acid sequence according to SEQ ID NO:91.

In certain embodiments, the antibody comprises a heavy chain (HC) amino acid sequence according to SEQ ID NO:92.

In certain embodiments, the antibody comprises a light chain (LC) amino acid sequence according to SEQ ID NO:93.

In certain embodiments, the antibody comprises a light chain (LC) amino acid sequence according to SEQ ID NO:94.

In certain embodiments, the antibody comprises an HC amino acid sequence according to SEQ ID NO: 91 and an LC amino acid sequence according to SEQ ID NO: 93.

In certain embodiments, the antibody comprises an HC amino acid sequence according to SEQ ID NO:92 and an LC amino acid sequence according to SEQ ID NO:94.

In certain embodiments, the antibody comprises an HC amino acid sequence according to SEQ ID NO:91 and an LC amino acid sequence according to SEQ ID NO:94.

In certain embodiments, the antibody comprises an HC amino acid sequence according to SEQ ID NO:92 and an LC amino acid sequence according to SEQ ID NO:93.

In some embodiments, the pharmaceutical compositions herein comprise (i) a heavy chain (HC) comprising an amino acid sequence according to SEQ ID NO: 91, and (ii) a light chain (LC) comprising an amino acid sequence according to SEQ ID NO: 93. (isolated), which binds to the antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus and hepatitis delta virus.

In some embodiments, the antibody binds to HBsAg of a genotype selected from HBsAg genotypes A, B, C, D, E, F, G, H, I, and J or any combination thereof. do.

In some embodiments, the antibody or pharmaceutical composition reduces serum levels of HBV DNA in a mammal with an HBV infection. In some embodiments, the antibody or pharmaceutical composition reduces serum levels of HBsAg in a mammal with an HBV infection. In some embodiments, the antibody or pharmaceutical composition reduces serum levels of HBeAg in a mammal with an HBV infection. In some embodiments, the antibody or pharmaceutical composition reduces serum levels of HBcrAg in a mammal with an HBV infection.

In some embodiments, the pharmaceutical compositions herein provide a heavy chain variable region (V _H ), and a light chain variable region (V _L ) comprising a CDRL1 amino acid sequence according to SEQ ID NO: 37, a CDRL2 amino acid sequence according to SEQ ID NO: 38 or 39, and a CDRL3 amino acid sequence according to SEQ ID NO: 58 or 40, and an Fc portion. , the Fc portion includes G236A/A330L/I332E.

In certain embodiments, the Fc portion does not include S239D. In certain embodiments, the Fc portion includes Ser (S) at position 239.

In certain embodiments, the Fc portion further comprises M428L/N434S.

In certain embodiments, the V _H comprises or consists of an amino acid sequence according to any one of SEQ ID NO: 41 or 67, and the V _L comprises an amino acid sequence according to any one of SEQ ID NO: 42, 59, 65, 89, 90, and 111-120. comprises or consists of an amino acid sequence according to any one of these.

In other embodiments, a heavy chain variable region (V _H ) comprising (i) a CDRH1 amino acid sequence according to SEQ ID NO:97, a CDRH2 amino acid sequence according to SEQ ID NO:98, a CDRH3 amino acid sequence according to SEQ ID NO:99, (ii) according to SEQ ID NO:100. A pharmaceutical composition comprising a light chain variable region (V _L ) comprising a CDRL1 amino acid sequence, a CDRL2 amino acid sequence according to SEQ ID NO: 100, and a CDRL3 amino acid sequence according to SEQ ID NO: 102, and (iii) an antibody or antigen-binding fragment thereof comprising an Fc portion. The Fc portion includes G236A/A330L/I332E.

In certain such embodiments of antibodies of pharmaceutical compositions, the V _H comprises or consists of the amino acid sequence according to SEQ ID NO:95 and the V _L comprises or consists of the amino acid sequence according to SEQ ID NO:96.

In certain embodiments, the Fc portion does not include S239D. In certain embodiments, the Fc portion further comprises M428L/N434S.

In some embodiments, the antibody of the pharmaceutical composition has enhanced binding to human FcγRIIA, human FcγRIIIA, or both as compared to a reference polypeptide that includes an Fc portion that does not include G236A/A330L/I332E. (wherein the human FcγRIIA may be H131 or R131, and/or the human FcγRIIIA may be F158 or V158), as compared to a reference polypeptide containing an Fc portion that does not include G236A/A330L/I332E. has reduced binding to human FcγRIIB and has reduced binding to human C1q compared to a reference polypeptide comprising an Fc portion that does not bind human FcγRIIB and does not include G236A/A330L/I332E; and activates FcγRIIA, human FcγRIIIA, or both to a greater extent than a reference polypeptide comprising an Fc portion that does not include G236A/A330L/I332E (where human FcγRIIA may be H131 or R131; and/or human FcγRIIIA may be F158 or V158), does not activate human FcγRIIB, and/or to a greater extent than a reference polypeptide containing an Fc portion that does not include G236A/A330L/I332E in the presence of HBsAg. activates human natural killer (NK) cells.

The pharmaceutical composition includes sufficient antibody material to facilitate administration of a therapeutically effective amount of the antibody to a patient. In some embodiments, the antibody is 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, and Contained at a concentration selected from 200 mg/mL. In other embodiments, the antibody is greater than 50 mg/mL, greater than 75 mg/mL, greater than 100 mg/mL, greater than 125 mg/mL, greater than 150 mg/mL, greater than 175 mg/mL, greater than 200 mg/mL, greater than 225 mg/mL, and 250 mg/mL. /mL in the composition. In other embodiments, the composition comprises the antibody at a concentration selected from the range of 50 mg/mL to 200 mg/mL, the range of 75 mg/mL to 225 mg/mL, and the range of 100 mg/mL to 200 mg/mL. In some embodiments, the composition includes the antibody at a concentration ranging from 125 mg/ml to 150 mg/ml. In yet other embodiments, the composition comprises the antibody at a concentration of 150 mg/mL.

Compositions according to the present invention include buffers, surfactants or triblock copolymers, salts (e.g., sodium chloride), and stabilizers [sugar alcohol, disaccharide, or polysaccharide stabilizers, and/or stabilizing amino acids (e.g., arginine). and/or glycine)]. Additionally, if necessary or desired, the compositions described herein can be formulated to further include one or more antioxidants (e.g., ascorbic acid, methionine, ethylenediaminetetraacetic acid (EDTA)). may be changed.

The pharmaceutical compositions of the present disclosure maintain antibody viability and exhibit and maintain a pH suitable for injection or infusion. The compositions described herein generally have a pH ranging from about 5.5 to about 8.5. In certain embodiments, the pharmaceutical composition has a pH in the range of about 5.5 to about 6.5, such as in the range of 5.5 to 6.5. In certain embodiments, the pharmaceutical composition has a pH in the range of 5.8 to 6.2, such as about 6.0. In certain embodiments, the pH is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or It may be 6.5. In some embodiments, the composition has a pH in the range of 6-8, such as about 7. In certain such embodiments, the pH may be about 6, such as 6.

The composition may include buffering agents to achieve and maintain the desired pH. Suitable buffers for use in the compositions described herein include, for example, acetate, citrate, histidine, succinate, phosphate, and hydroxymethylaminomethane (Tris) buffers. included. In certain embodiments, the composition includes a buffer selected from a histidine buffer and a phosphate buffer. In certain embodiments, the composition exhibits a pH of 6 and includes a histidine buffer. In such embodiments, histidine may be included in the composition at a concentration ranging from 10mM to 40mM (eg, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, or 40mM). For example, in certain embodiments, compositions according to the present invention exhibit a pH of 6 and include histidine at a concentration selected from 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, and 40 mM.

Pharmaceutical compositions described herein may include a surfactant or a triblock copolymer. Surfactants, sometimes referred to as "detergents", can serve one or more functions. For example, in aqueous antibody solutions, surfactants serve to preserve the functionality of the antibody, assist in solubilizing the antibody or other excipients, and/or control microbial growth. Surfactants used in the compositions described herein include, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20). Additionally or alternatively, triblock copolymers such as poloxamer 188 may also be used. In some embodiments, the composition includes a surfactant at a concentration ranging from 0.01% to 0.05% (w/v). In such embodiments, the surfactant may be selected from polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188. In certain embodiments, the pharmaceutical compositions herein include polysorbate 80 (Tween 80) at a concentration ranging from 0.01% to 0.05% (w/v). In other embodiments, the pharmaceutical compositions herein include polysorbate 80 (Tween 80) at a concentration of 0.02% (w/v).

When the composition according to the present disclosure includes a sugar alcohol, disaccharide, or polysaccharide stabilizer, the stabilizer may be selected from, for example, mannitol, sorbitol, sucrose, trehalose, and dextran-40. In certain embodiments, the stabilizer is a disaccharide. In certain embodiments, the pharmaceutical composition comprises a disaccharide at a concentration selected from 4.0% to 10% (w/v). In certain such embodiments, the disaccharide is sucrose. In other embodiments, the pharmaceutical composition is 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, Contains sucrose at a concentration selected from 9.8%, 9.9%, or 10.0% (w/v), or within a range bounded and inclusive of any two of these values. In yet other embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 7%, such as 7% (w/v).

In some embodiments, the composition is adapted for administration to a mammal, such as a human. In such embodiments, the composition is specifically formulated to be sterile and pyrogen-free. Additionally, the composition may be isotonic to humans.

The compositions described herein may be prepared for direct administration to a subject (i.e., without reconstitution or mixing steps) or reconstituted in an aqueous medium prior to injection or infusion into a patient. It may also be prepared as a lyophilized material consisting of: For direct administration to a subject, pharmaceutical compositions according to the present disclosure may be provided in a vial, such as, for example, a prefilled syringe or a glass vial. In some embodiments, the pharmaceutical compositions of the present disclosure are supplied in a hermetically sealed container. In some embodiments, the composition may be in the form of a kit designed to be reconstituted immediately before the combined composition is administered to a subject. For example, lyophilized antibodies may be provided in a kit with sterile water or sterile buffer.

Administration of pharmaceutical compositions according to the present disclosure in methods and uses according to the present disclosure can be carried out alone or in combination with co-agents (also referred to herein as "further active ingredients"), which may be associated with hepatitis B Useful for preventing and/or treating viral infections.

The present disclosure encompasses the administration of a pharmaceutical composition according to the present disclosure, wherein the pharmaceutical composition is administered prior to a co-agent or another therapeutic regimen useful for treating and/or preventing hepatitis B virus infection. administered to the subject at the same time or subsequently. The pharmaceutical composition administered in combination with the co-agent may be administered in the same or different compositions and by the same or different routes of administration. As used herein, expressions such as "combination therapy," "combined administration," "administered in combination" and the like refer to the combined action of drugs (administered "in combination"). To this end, the combined drugs are usually present at the site of action at the same time and/or in overlapping time windows. While the effects caused by one of the drugs are still ongoing (even though the drug itself is no longer present at detectable levels), the other drug is administered, thereby reciprocating the effects of both drugs. It is also possible that it can act. However, it may have been administered long before the other drug (e.g., more than 1, 2, or 3 months or 1 year ago) and is no longer present at detectable levels (or its effects persist) by the time the other drug is administered. drugs (not administered) are typically not considered to have been administered "in combination."

In certain embodiments, the pharmaceutical compositions of the present disclosure contain a PD-1 inhibitor, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP-224, BMS-936558, or any of the foregoing. used in combination with PD-1 specific antibodies or binding fragments thereof, such as combinations.

In certain embodiments, the pharmaceutical compositions of the present disclosure include PD-L1-specific antibodies, such as BMS-936559, duvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof. or in combination with binding fragments thereof.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of CTLA4. In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with CTLA4-specific antibodies or binding fragments thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof. used.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a B7-H3-specific antibody or binding fragment thereof, such as enobrituzumab (MGA271), 376.96, or both. B7-H3 antibody-binding fragments are, for example, scFvs described in Dangaj et al., Cancer Res. 73:4820, 2013, and in US Pat. or a fusion protein thereof.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of CD244.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT publication WO2010/084158.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of TIM3.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of Gal9.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of A2aR.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of KIR, such as rililumab (BMS-986015).

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitory cytokine (typically a cytokine other than TGFβ) or an inhibitor of Treg development or activity.

In certain embodiments, the pharmaceutical compositions of the present disclosure contain levo-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), and ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (1-MT)-thira-pazamine , or any combination thereof.

In certain embodiments, the pharmaceutical compositions of the present disclosure include N(omega)-nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-1-arginine (nor-NOHA), used in combination with an arginase inhibitor such as L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof. Ru.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

In certain embodiments, the pharmaceutical compositions of the present disclosure are combined with an inhibitor of TIGIT, such as, for example, COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as, for example, COM701 (Compugen), or both. used.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT publication WO2016/134333. Anti-PVRL2 antibodies are described, for example, in PCT publication WO2017/021526.

In certain embodiments, compositions of the present disclosure are used in combination with LAIR1 inhibitors.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with agents that increase the activity of stimulatory immune checkpoint molecules (ie, are agonists). For example, compositions of the present disclosure may include CD137 (4-1BB) agonists (e.g., urelumab), CD134 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, CD27 agonists (e.g., CDX-1127), ), CD28 agonists (e.g. TGN1412, CD80, or CD86), CD40 agonists (e.g. CP-870,893, rhuCD40L, or SGN-40), CD122 agonists (e.g. IL-2), agonists of GITR (e.g. PCT Patent Publication WO 2016) /054638), in combination with an agonist of ICOS (CD278) (e.g. GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos 314-8, or any combination thereof) can be used. In any of the embodiments disclosed herein, the method comprises administering a pharmaceutical composition of the present disclosure to one or more agonists of stimulatory immune checkpoint molecules, including any of the above alone or in any combination. may include administration together.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a nucleoside reverse transcriptase inhibitor (NRTI), an interferon (e.g., IFNα, IFNβ, or both), or any combination thereof. be done. In some embodiments, the NRTI is one of tenofovir, tenofovir disoproxil (e.g., tenofovir disoproxil fumarate), tenofovir alafenamide, entecavir, lamivudine, adefovir, and adefovir dipivoxil. or more than one.

Medical Treatment and Uses In a further aspect, the disclosure provides the use of a pharmaceutical composition according to the disclosure in the treatment of infection by hepatitis B virus. In certain embodiments, the present disclosure provides methods for the treatment of infection by hepatitis B virus, including methods comprising administering a therapeutically effective amount of a pharmaceutical composition according to the present disclosure to a subject in need thereof. I will provide a.

In a therapeutic setting, the subject is infected with a hepatitis B virus infection, has been diagnosed with a hepatitis B virus infection, and/or is exhibiting symptoms of a hepatitis B virus infection. Of note, the terms "treatment" and "therapy/therapy" for hepatitis B virus refer to (complete) cure and attenuation/reduction of hepatitis B virus infection and/or associated symptoms (e.g. infection and/or treatment). or severity of symptoms, number of symptoms, duration of infection and/or symptoms, or attenuation/reduction of any combination thereof).

In certain embodiments, the subject is an adult. In certain embodiments, the subject ranges in age from 18 to 65 years old. In certain embodiments, the subject's weight is between 40 kg and 125 kg. In certain embodiments, the subject has a body mass index (BMI) of 18-35 kg/m ² .

In certain embodiments, the subject receiving the pharmaceutical compositions of the present disclosure has chronic disease, e.g., as defined by positive sera for HBsAg, HBV DNA, and/or HBeAg on two occasions at least 6 months apart. Have HBV infection.

In certain embodiments, the subject receiving the pharmaceutical composition of the present disclosure does not have cirrhosis. The absence of cirrhosis is determined by Fibroscan evaluation (e.g. within 6 months prior to administration of a single dose of the pharmaceutical composition) or liver biopsy (e.g. within 12 months prior to administration of a single dose of the pharmaceutical composition). Preferably, the absence of cirrhosis is determined by the absence of Metavir F3 fibrosis or the absence of F4 cirrhosis.

In certain embodiments, a subject receiving a pharmaceutical composition of the present disclosure receives a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI), optionally for 120 hours, before a single dose of the pharmaceutical composition is administered. within 60 days, as appropriate. In other words, the subject has already received an NRTI, such as within 120 days or within 60 days of administration of the pharmaceutical composition.

In certain embodiments, the NRTI is one of tenofovir, tenofovir disoproxil (e.g., tenofovir disoproxil fumarate), tenofovir alafenamide, entecavir, lamivudine, adefovir, and adefovir dipivoxil. Contains one or more.

In certain embodiments, a subject receiving a pharmaceutical composition of the present disclosure receives 100 IU/mL (e.g., 99, 98, 97, 96, 95, 90, 80, 70, 60, etc.).

In certain embodiments, a subject receiving a pharmaceutical composition of the present disclosure has a serum HBsAg concentration of less than 3,000 IU/mL before a single dose is administered. In certain embodiments, a subject receiving a pharmaceutical composition of the present disclosure has a serum HBsAg concentration of less than 1,000 IU/mL before a single dose is administered.

In certain embodiments, a subject receiving a pharmaceutical composition of the present disclosure has a serum HB surface antigen (HBsAg) concentration of 3,000 IU/mL or greater for a period of no more than 28 days before the single dose is administered. has. In certain embodiments, a subject receiving a pharmaceutical composition of the present disclosure has a serum HB surface antigen (HBsAg) concentration of 1,000 IU/mL or greater for a period of no more than 28 days before the single dose is administered. has. HBsAg concentration can be determined using, for example, the Abbott ARCHITECT assay.

In certain embodiments, the subject receiving a pharmaceutical composition of the present disclosure has been HBe antigen (HBeAg) negative for no more than 28 days before the single dose is administered.

In certain embodiments, the subject was negative for anti-HB antibodies for no more than 28 days before the single dose was administered.

In certain embodiments, a subject receiving a pharmaceutical composition of the present disclosure is
(i) does not have fibrosis and/or does not have cirrhosis; and/or (ii) has (serum) alanine aminotransferase (ALT) lower than 2 times the upper limit of normal (ULN).

In certain embodiments, the method comprises administering a single dose of a pharmaceutical composition of the present disclosure.

In some embodiments, a single dose of the pharmaceutical composition is in the range of 2-18 mg/kg (subject's body weight), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 mg/kg of antibody.

In certain embodiments, a single dose of the pharmaceutical composition comprises up to 6 mg, up to 18 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of antibody. In certain embodiments, a single dose of the pharmaceutical composition comprises about 10, about 25, about 50, about 75, about 90, about 100, about 125, about 150, about 175, about 200, about 250, about 300 , about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1250, about 1500, about 1750, about 2000, about 2250, about 2500, about 2750, or about 3000 mg of antibody.

In certain embodiments, a single dose of the pharmaceutical composition is 6 mg, up to 10 mg, up to 15 mg, up to 18 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 mg. , up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody.

In certain embodiments, a single dose of the pharmaceutical composition is in the range of 1 mg to 3000 mg, or in the range of 5 mg to 3000 mg, or in the range of 6 mg to 3000 mg, or in the range of 10 mg to 3000 mg, or in the range of 25 mg to 3000 mg, or in the range of 30 mg to In the range 3000mg, or in the range 50mg to 3000mg, or in the range 60mg to 3000mg, or in the range 75mg to 3000mg, or in the range 90mg to 3000mg, or in the range 100mg to 3000mg, or in the range 150mg to 3000mg, or in the range 200mg to 3000mg or 300mg to 3000mg, or 400mg to 3000mg, or 500mg to 3000mg, or 600mg to 3000mg, or 750mg to 3000mg, or 900mg to 3000mg, or 1000mg to 3000mg. or from 1500 mg to 3000 mg, or from 2000 mg to 3000 mg.

In certain embodiments, a single dose of the pharmaceutical composition is in the range 1 mg to 900 mg, or in the range 5 mg to 900 mg, or in the range 6 mg to 900 mg, in the range 10 mg to 900 mg, in the range 25 mg to 900 mg, in the range 30 mg to 900mg range, 50mg to 900mg range, 60mg to 900mg range, 75mg to 900mg range, 90mg to 900mg range, 100mg to 900mg range, 150mg to 900mg range, 200mg to 900mg range, 300mg to 900mg range range, 500 mg to 900 mg, 750 mg to 900 mg.

In certain embodiments, a single dose of the pharmaceutical composition is in the range 1 mg to 500 mg, or in the range 5 mg to 500 mg, or in the range 6 mg to 500 mg, in the range 10 mg to 500 mg, in the range 25 mg to 500 mg, in the range 30 mg to 500mg range, 50mg to 500mg range, 60mg to 500mg range, 75mg to 500mg range, 90mg to 500mg range, 100mg to 500mg range, 150mg to 500mg range, 200mg to 500mg range, 300mg to 500mg range range, including antibodies in amounts ranging from 300 mg to 500 mg.

In certain embodiments, a single dose of the pharmaceutical composition is in the range 1 mg to 300 mg, or in the range 5 mg to 300 mg, or in the range 6 mg to 300 mg, in the range 10 mg to 300 mg, in the range 25 mg to 300 mg, in the range 30 mg to antibody in amounts ranging from 300 mg to 300 mg, from 60 mg to 300 mg, from 75 mg to 300 mg, from 90 mg to 300 mg, from 100 mg to 300 mg, from 150 mg to 300 mg, from 200 mg to 300 mg. include.

In certain embodiments, a single dose of the pharmaceutical composition is in the range 1 mg to 200 mg, or in the range 5 mg to 200 mg, or in the range 6 mg to 200 mg, in the range 10 mg to 200 mg, in the range 25 mg to 200 mg, in the range 30 mg to 200 mg to 200 mg, 60 mg to 200 mg, 75 mg to 200 mg, 90 mg to 200 mg, 100 mg to 200 mg, 150 mg to 200 mg.

In certain embodiments, a single dose of the pharmaceutical composition is in the range 1 mg to 100 mg, or in the range 5 mg to 100 mg, or in the range 6 mg to 100 mg, in the range 10 mg to 100 mg, in the range 25 mg to 100 mg, in the range 30 mg to including antibodies in amounts ranging from 100 mg, from 60 mg to 100 mg, from 75 mg to 100 mg, from 90 mg to 100 mg.

In certain embodiments, a single dose of the pharmaceutical composition is in the range 1 mg to 25 mg, or in the range 5 mg to 25 mg, or in the range 6 mg to 25 mg, or in the range 10 mg to 25 mg, or in the range 15 mg to 25 mg, or the antibody in an amount ranging from 20 mg to 25 mg.

In certain embodiments, a single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 1 mg to 15 mg, or from 5 mg to 15 mg, or from 6 mg to 15 mg, or from 10 mg to 15 mg.

In certain embodiments, the single dose pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885,890,895,900,905,910,915,920,925,930,935,940,945,950,955,960,965,970,975,980,985,990,995, or 1000mg, or Contains more antibodies.

In certain embodiments, a single dose of the pharmaceutical composition is less than 3000 mg, less than 2500 mg, less than 2000 mg, less than 1500 mg, less than 1000 mg, less than 900 mg, less than 500 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 90 mg, less than 75 mg. the antibody in an amount less than, less than 50 mg, less than 25 mg, or less than 10 mg, but greater than or equal to 1 mg, greater than or equal to 2 mg, greater than or equal to 3 mg, greater than or equal to 4 mg, greater than or equal to 5 mg, or greater than or equal to 6 mg.

In certain embodiments, a single dose of the pharmaceutical composition contains about 75 mg of antibody. In other embodiments, a single dose of the pharmaceutical composition contains about 90 mg of antibody. In yet other embodiments, a single dose of the pharmaceutical composition contains up to 300 mg of antibody. In yet other embodiments, a single dose of the pharmaceutical composition contains up to 900 mg of antibody. In yet other embodiments, a single dose of the pharmaceutical composition contains up to 3,000 mg of antibody.

In certain embodiments, single dose pharmaceutical compositions range from 100 mg/mL to 200 mg/mL, such as 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL. , 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, or 200 mg/mL, preferably 150 mg/mL.

In any of the methods described herein for the treatment of infection by hepatitis B virus, the pharmaceutical composition can be administered by injection or infusion. When administered by infusion, the pharmaceutical composition may be administered, for example, by intravenous, intraarterial, or intraventricular injection. When administered by injection, the pharmaceutical composition may be administered, for example, by intravenous, intraarterial, intraventricular, intramedullary, intraperitoneal, intrathecal, intraventricular, or subcutaneous injection. In certain embodiments of the methods described herein, the pharmaceutical composition is administered by subcutaneous ("SC") or intravenous ("IV") injection.

A dose is referred to as a "single dose" and an administration is considered a "single dose" even if multiple injections or infusions are required to administer a defined dose. Generally, when multiple injections or infusions are required to administer a single defined dose, the multiple injections or infusions will be about 5 minutes or less, about 15 minutes or less, about 30 minutes or less, about The administration is administered for a period of less than 1 hour, less than about 2 hours, less than about 4 hours, less than about 6 hours, less than about 1 day, less than about 1 week, or less than about 1 month.

In certain embodiments, on about 56 days after administration of the single dose, the subject has a serum HBsAg of less than one-half compared to the subject's serum HBsAg on days 0 to 28 prior to administration of the single dose. have a decrease in serum HBsAg (e.g., the concentration of serum HBsAg determined using the Abbott ARCHITECT assay) to

In certain embodiments, after administration of a single dose of the pharmaceutical composition (e.g., on day 56 after administration of the single dose), the subject (i) has received a placebo or treatment for HBV for the same period of time; reduced compared to a reference subject who did not receive the drug (e.g., a subject with a similar severity of HBV infection and of the same sex, age, weight, and/or general health status as the subject receiving the pharmaceutical composition) (ii) have an adaptive immune response to HBV, including, for example, an HBV-specific T cell response.

In some embodiments, after administration of a single dose, the subject's serum HBsAg decreases by 1.0 log ¹⁰ IU/mL, 1.5 log ¹⁰ IU/mL, or more compared to baseline (pre-dose). However, the reduction may persist for 1, 2, 3, 4, 5, 6, 7, 8, or more days after administration of a single dose. In some embodiments, a single dose contains 6 mg of antibody or more. In some embodiments, after administration of the single dose, the subject's serum HBsAg is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, Decreases over 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 days or more.

In certain embodiments, after administration of a single dose, the subject's serum HBsAg is reduced for at least 8 days, at least 15 days, at least 22 days, or at least 29 days compared to baseline (before administration).

In certain embodiments, at least 100 ng/mL of the antibody remains in serum for at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days after administration of a single dose. It remains unbound to HBsAg. In certain embodiments, at least 100 ng/mL of the antibody is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, Remains unbound to serum HBsAg for 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 days or more. In certain embodiments, at least 100 ng/mL of the antibody remains unbound to serum HBsAg for at least 8 days after administration of a single dose.

In certain embodiments, at least 1000 ng/mL of the antibody is at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, It remains unbound to serum HBsAg for 28 days or more. In certain embodiments, at least 1000 ng/mL of the antibody remains unbound to serum HBsAg for at least 14 days after administration of a single dose.

In certain embodiments, the C _max of the concentration of antibody in the subject is between 300 ng/mL and 6,000 ng/mL after administration of a single dose. In some embodiments, the C _max of the concentration of the antibody in the subject is at least 300 ng/mL, 400 ng/mL, 500 ng/mL, 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1000 ng/mL, 1100ng/mL, 1200ng/mL, 1300ng/mL, 1400ng/mL, 1500ng/mL, 1600ng/mL, 1700ng/mL, 1800ng/mL, 1900ng/mL, 2000ng/mL, 2100ng/mL, 2200ng/mL, 230 0ng/ ML, 2400 ng / ml, 2500 ng / ml, 2600 ng / ml, 2700 ng / ml, 2800 ng / ml, 2900 ng / ml, 300 ng / ml, 3100 ng / ml, 3200 ng / ml, 3300 ng / ml, 3300 ng / ml, 3300 ng / ml. 400 ng / ml, 3500 ng / ml, 3600ng/mL, 3700ng/mL, 3800ng/mL, 3900ng/mL, 4000ng/mL, 4100ng/mL, 4200ng/mL, 4300ng/mL, 4400ng/mL, 4500ng/mL, 4600ng/mL, 4700ng/mL, 480 0ng/ ML, 4900 ng / ml, 5000 ng / ml, 5100 ng / ml, 5200 ng / ml, 5300 ng / ml, 5400 ng / ml, 5400 ng / ml, 5500 ng / ml, 5600 ng / ml, 5700 ng / ml, 580 0 ng / ml, 5900 ng / ml, 6000 ng / ml be.

This disclosure also includes the following exemplary embodiments.

Embodiment 1. 91. A method of treating hepatitis B virus (HBV) infection in a subject, said method comprising administering to said subject a single dose of a pharmaceutical composition comprising an antibody, said antibody comprising: a heavy chain amino acid sequence and a light chain amino acid sequence of SEQ ID NO: 93; (a) said single dose pharmaceutical composition comprises at least 6 mg of said antibody;
(b) after administration of the single dose, the subject's serum HBsAg decreases by at least 1.0 log ¹⁰ IU/mL, 1.5 log ¹⁰ IU/mL, or more compared to baseline;
(c) the reduction in serum HBsAg in said subject persists for 1, 2, 3, 4, 5, 6, 7, 8, or more days after administration of said single dose.
Embodiment 2. 91. A method of treating hepatitis B virus (HBV) infection in a subject, said method comprising administering to said subject a single dose of a pharmaceutical composition comprising an antibody, said antibody comprising: comprising a heavy chain amino acid sequence and a light chain amino acid sequence of SEQ ID NO: 93;
(a) said single dose pharmaceutical composition comprises at least 75 mg of said antibody;
(b) after administration of the single dose, the subject's serum HBsAg decreases by at least 1.0 log ¹⁰ IU/mL, at least 1.5 log ¹⁰ IU/mL, or more compared to baseline;
(c) said subject's serum HBsAg decreases at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 after administration of said single dose; A method lasting for 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 days or more.
Embodiment 3. A method of treating hepatitis B virus (HBV) infection in a subject, said method comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody;
(a) said antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 91 and a light chain amino acid sequence of SEQ ID NO: 93; and (b) at least 1000 ng/mL of the antibody is present for at least 14 days after administration of said single dose. remains unbound to serum HBsAg,
(c) the subject has a baseline serum HBsAg level of less than 3000 IU/mL.
Embodiment 4. 4. The method of any one of embodiments 1-3, wherein the subject's serum HBsAg decreases by at least 1.0 log ¹⁰ IU/mL within 8 days of administration of a single dose compared to baseline.
Embodiment 5. said single dose of the pharmaceutical composition comprises at least 75 mg of said antibody, and said subject's serum HBsAg is at least 1.5 log ¹⁰ IU/dose within 8 days of administration of said single dose as compared to baseline. 5. The method of any one of embodiments 1-4, wherein the mL is reduced.
Embodiment 6. The method of any one of embodiments 1-5, wherein the subject's serum HBsAg is reduced by at least 0.5 log ¹⁰ IU/mL on day 56 after administration of the single dose compared to baseline. .
Embodiment 7. 7. The method of any one of embodiments 1-6, wherein the subject has a C _max for the antibody between 300 ng/mL and 6,000 ng/mL.
Embodiment 8. C _max of the concentration of the antibody in the subject is at least 300ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL, 1000ng/mL, 1100ng/mL, 1200ng / Ml, 1300 ng / ml, 1400 ng / ml, 1500 ng / ml, 1600 ng / ml, 1700 ng / ml, 1800 ng / ml, 1700 ng / ml, 2000 ng / ml, 2000 ng / ml, 2100 ng / ml, 2200 ng / ml , 2300 ng / ml, 2400 ng / ml , 2500ng/mL, 2600ng/mL, 2700ng/mL, 2800ng/mL, 2900ng/mL, 3000ng/mL, 3100ng/mL, 3200ng/mL, 3300ng/mL, 3400ng/mL, 3500ng/mL, 3600ng/mL, 37 00ng / Ml, 3800 ng / ml, 3900 ng / ml, 4000 ng / ml, 4100 ng / ml, 4200 ng / ml, 4300 ng / ml, 4400 ng / ml, 4500 ng / ml, 4600 ng / ml, 470 0 ng / ml, 4800 ng / ml, 4900 ng / ml , 5000ng/mL, 5100ng/mL, 5200ng/mL, 5300ng/mL, 5400ng/mL, 5500ng/mL, 5600ng/mL, 5700ng/mL, 5800ng/mL, or 5900ng/mL, according to embodiment 7. Method.
Embodiment 9. The single dose of the pharmaceutical composition may be up to 10 mg, up to 15 mg, up to 18 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 mg, up to 75 mg, up to 90 mg, up to 300 mg. up to 900 mg, or up to 3000 mg of the antibody,
or said single dose of the pharmaceutical composition is in the range 6 mg to 3000 mg, or in the range 10 mg to 3000 mg, or in the range 25 mg to 3000 mg, in the range 30 mg to 3000 mg, in the range 50 mg to 3000 mg, in the range 60 mg to 3000 mg, in the range 75 mg ~3000mg range, 90mg~3000mg range, 100mg~3000mg range, 150mg~3000mg range, 200mg~3000mg range, 300mg~3000mg range, 500mg~3000mg range, 750mg~3000mg range, 900mg~3000 mg 1500 mg to 3000 mg, 2000 mg to 3000 mg,
or said single dose of the pharmaceutical composition is in the range 6 mg to 900 mg, or in the range 10 mg to 900 mg, or in the range 25 mg to 900 mg, in the range 30 mg to 900 mg, in the range 50 mg to 900 mg, in the range 60 mg to 900 mg, in the range 75 mg. 900mg to 900mg, 90mg to 900mg, 100mg to 900mg, 150mg to 900mg, 200mg to 900mg, 300mg to 900mg, 500mg to 900mg, 750mg to 900mg. Contains antibodies;
or said single dose of the pharmaceutical composition is in the range 6 mg to 500 mg, or in the range 10 mg to 500 mg, or in the range 25 mg to 500 mg, in the range 30 mg to 500 mg, in the range 50 mg to 500 mg, in the range 60 mg to 500 mg, in the range 75 mg. 500 mg, 90 mg to 500 mg, 100 mg to 500 mg, 150 mg to 500 mg, 200 mg to 500 mg, 300 mg to 500 mg, 400 mg to 500 mg,
or said single dose of the pharmaceutical composition is in the range 6 mg to 300 mg, or in the range 10 mg to 300 mg, or in the range 25 mg to 300 mg, in the range 30 mg to 300 mg, in the range 50 mg to 300 mg, in the range 60 mg to 300 mg, in the range 75 mg. 300 mg, 90 mg to 300 mg, 100 mg to 300 mg, 150 mg to 300 mg, 200 mg to 300 mg,
or said single dose of the pharmaceutical composition is in the range 6 mg to 200 mg, or in the range 10 mg to 200 mg, or in the range 25 mg to 200 mg, in the range 30 mg to 200 mg, in the range 50 mg to 200 mg, in the range 60 mg to 200 mg, in the range 75 mg. 200 mg, 90 mg to 200 mg, 100 mg to 200 mg, 150 mg to 200 mg,
or said single dose of the pharmaceutical composition is in the range 6 mg to 100 mg, or in the range 10 mg to 100 mg, or in the range 25 mg to 100 mg, in the range 30 mg to 100 mg, in the range 50 mg to 100 mg, in the range 60 mg to 100 mg, in the range 75 mg. -100 mg, 75 mg to 100 mg, 90 mg to 100 mg, or said single dose pharmaceutical composition comprises 6 mg to 25 mg, or 10 mg to 25 mg, or 15 mg. comprising said antibody in an amount in the range of ~25 mg, or in the range of 20 mg to 25 mg;
or said single dose of the pharmaceutical composition is in the range 6 mg to 50 mg, or in the range 6 mg to 25 mg, or in the range 6 mg to 50 mg, or in the range 10 to 50 mg, or in the range 10 to 25 mg, or in the range 6 mg to 15 mg. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130 , 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255 , 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380 , 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505 , 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630 , 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755 , 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880 , 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 mg, or more of the above-mentioned antibodies,
or the single dose of the pharmaceutical composition is less than 3000 mg, less than 2500 mg, less than 2000 mg, less than 1500 mg, less than 1000 mg, less than 900 mg, less than 500 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 90 mg, less than 75 mg, less than 50 mg, 9. The method of any one of embodiments 1-8, comprising said antibody in an amount of less than 25 mg, or less than 10 mg.
Embodiment 10. The single dose of the pharmaceutical composition may be in the range of 100 mg/mL to 200 mg/mL, such as 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg. 10. The method according to any one of embodiments 1 to 9, comprising said antibody at a concentration of 180 mg/mL, 180 mg/mL, 190 mg/mL or 200 mg/mL, preferably 150 mg/mL.
Embodiment 11. 11. The method of any one of embodiments 1-10, wherein the single dose of the pharmaceutical composition comprises about 75 mg of the antibody.
Embodiment 12. 11. The method of any one of embodiments 1-10, wherein the single dose of the pharmaceutical composition comprises about 90 mg of the antibody.
Embodiment 13. 11. The method of any one of embodiments 1-10, wherein the single dose of the pharmaceutical composition comprises up to 300 mg of the antibody.
Embodiment 14. 11. The method of any one of embodiments 1-10, wherein the single dose of the pharmaceutical composition comprises up to 900 mg of the antibody.
Embodiment 15. 11. The method of any one of embodiments 1-10, wherein the single dose of the pharmaceutical composition comprises up to 3,000 mg of the antibody.
Embodiment 16. The method comprises administering the single dose by subcutaneous injection, and the single dose may comprise or consist of 6 mg of the antibody, 18 mg of the antibody, or 75 mg of the antibody. , the method of any one of embodiments 1-15.
Embodiment 17. 17. The method of any one of embodiments 1-16, comprising administering said single dose by intravenous injection.
Embodiment 18. 18. The method according to any one of embodiments 1-17, wherein the pharmaceutical composition further comprises water, optionally water.
Embodiment 19. 19. A method according to any one of embodiments 1 to 18, wherein the pharmaceutical composition further comprises histidine, optionally at a concentration in the range of 10mM to 40mM, such as 20mM.
Embodiment 20. Embodiments 1 to 19, wherein said pharmaceutical composition further comprises a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8% or 9%, preferably about 7% (w/v). The method described in any one of the above.
Embodiment 21. The pharmaceutical composition further comprises a surfactant or a triblock copolymer, optionally a polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), wherein the polysorbate or poloxamer 188 is optionally comprised between 0.01% and 0.05%. % (w/v), preferably 0.02% (w/v).
Embodiment 22. The pharmaceutical composition has a pH in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or in the range of 5.8, 5.9, 6.0, 6.1, or 6.2. 22. The method according to any one of embodiments 1-21, comprising:
Embodiment 23. The pharmaceutical composition comprises:
(i) 150 mg/mL of said antibody;
(ii) USP Water;
(iii) 20mM histidine;
(iv) 7% sucrose, and (v) 0.02% PS80.
and the pH is 6.
Embodiment 24. 24. The method of any one of embodiments 1-23, wherein the subject is an adult.
Embodiment 25. 25. The method of embodiment 24, wherein the subject ranges in age from 18 to 65 years.
Embodiment 26. 26. The method of any one of embodiments 1-25, wherein the subject has a body weight of 40 kg to 125 kg and/or the subject has a body mass index (BMI) of 18 to 35 kg/ ^m2 .
Embodiment 27. Embodiment 1, wherein the subject has chronic HBV infection, e.g., defined by positive sera for HBsAg, HBV DNA, and/or HBeAg on two occasions, said two occasions being at least 6 months apart. 27. The method according to any one of 26 to 26.
Embodiment 28. 28. The method of any one of embodiments 1-27, wherein the subject does not have cirrhosis.
Embodiment 29. The absence of cirrhosis is determined by Fibroscan evaluation (e.g. within 6 months prior to administration of said single dose of the pharmaceutical composition) or liver biopsy (e.g. within 12 months prior to administration of said single dose pharmaceutical composition). 29. The method according to embodiment 28, wherein the absence of cirrhosis is determined by the absence of Metavir F3 fibrosis or the absence of F4 cirrhosis.
Embodiment 30. Embodiments 1-29, wherein the subject has received a nucleoside reverse transcriptase inhibitor (NRTI) within 120 days, and even 60 days, before the single dose is administered. The method described in any one of the above.
Embodiment 31. the NRTI comprises one or more of tenofovir, tenofovir disoproxil (e.g., tenofovir disoproxil fumarate), tenofovir alafenamide, entecavir, lamivudine, adefovir, and adefovir dipivoxil; 31. The method of embodiment 30.
Embodiment 32. 32. The method of any one of embodiments 1-31, wherein the subject has a serum HBV DNA concentration of less than 100 IU/mL for a period of no more than 28 days before the single dose is administered.
Embodiment 33. said subject has a serum HBsAg concentration of less than 3,000 IU/mL before said single dose is administered and has a serum HBsAg concentration of less than 1,000 IU/mL before said single dose is administered; 33. The method according to any one of embodiments 1-32.
Embodiment 34. 33. The method according to any one of embodiments 1-32. said subject has a serum HBsAg concentration of 3,000 IU/mL or more for a period of no more than 28 days before said single dose is administered, and for a period of no more than 28 days before said single dose is administered; May have a serum HBsAg concentration of 1,000 IU/mL or higher.
Embodiment 35. 35. The method of any one of embodiments 1-34, wherein the subject was HBe antigen (HBeAg) negative for no more than 28 days before the single dose was administered.
Embodiment 36. 36. The method of any one of embodiments 1-35, wherein the subject was negative for anti-HB antibodies for no more than 28 days before the single dose was administered.
Embodiment 37. said subject, before said single dose is administered, (i) does not have fibrosis and/or does not have cirrhosis, and/or (ii) is below twice the upper limit of normal (ULN). 37. The method of any one of embodiments 1-36, wherein the method comprises alanine aminotransferase (ALT).
Embodiment 38. On day 56 after administration of said single dose, said subject has reduced serum HBsAg to less than half of said subject's serum HBsAg from day 0 to day 28 prior to administration of said single dose. 38. The method of any one of embodiments 1-37, wherein the method has a decrease in serum HBsAg concentration as determined using the Abbott ARCHITECT assay.
Embodiment 39. after administration of said single dose (e.g., on day 56 after administration of said single dose), said subject (i) has reduced or less severe intrahepatic spread of HBV compared to a reference subject; and/or (ii) an adaptive immune response against HBV.
Embodiment 40. 40. The method of any one of embodiments 1-39, wherein the subject is male.
Embodiment 41. 40. The method of any one of embodiments 1-39, wherein the subject is female.
Embodiment 42. A pharmaceutical composition comprising an antibody, wherein the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 91 and a light chain amino acid sequence of SEQ ID NO: 93, wherein the pharmaceutical composition comprises an antibody in the range of 100 mg/mL to 200 mg/mL; For example, 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, or 200 mg/mL, preferably 150 mg/mL. mL, and immediately after administering said composition to a subject in need thereof, at least 1000 ng/mL of the antibody remains unbound to serum HBsAg for at least 14 days after administration of said single dose. , wherein said subject has a baseline serum HBsAg level of less than 3000 IU/mL.
Embodiment 43. The pharmaceutical composition of Embodiment 42, comprising up to 6 mg, up to 18 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of said antibody.
Embodiment 44. 44. A pharmaceutical composition according to embodiment 42 or 43, comprising about 75 mg of said antibody.
Embodiment 45. 44. A pharmaceutical composition according to embodiment 42 or 43, comprising about 90 mg of said antibody.
Embodiment 46. 44. A pharmaceutical composition according to embodiment 42 or 43, comprising about 300 mg of said antibody.
Embodiment 47. 44. A pharmaceutical composition according to embodiment 42 or 43, comprising about 900 mg of said antibody.
Embodiment 48. 44. A pharmaceutical composition according to embodiment 42 or 43, comprising about 3,000 mg of said antibody.
Embodiment 49. 49. The pharmaceutical composition according to any one of embodiments 42-48, further comprising water, optionally USP water.
Embodiment 50. A pharmaceutical composition according to any one of embodiments 42 to 49, further comprising histidine optionally at a concentration of 10mM to 40mM, such as 20mM.
Embodiment 51.5%. Pharmaceutical composition according to any one of embodiments 42 to 50, further comprising a disaccharide, such as sucrose, which may be 6%, 7%, 8% or 9%, preferably about 7% (w/v). .
Embodiment 52. It further comprises a surfactant, optionally a polysorbate, preferably polysorbate 80 (PS80), wherein said polysorbate is optionally in the range of 0.01% to 0.05% (w/v), preferably 0.02% ( 52. The pharmaceutical composition according to any one of embodiments 42 to 51, wherein the pharmaceutical composition is present in w/v).
Embodiment 53. has a pH in the range 5.8 to 6.2, in the range 5.9 to 6.1, or in the range 5.8, 5.9, 6.0, 6.1, or 6.2. 53. A pharmaceutical composition according to any one of embodiments 42-52.
Embodiment 54. (i) 150 mg/mL of said antibody;
(ii) USP Water;
(iii) 20mM histidine;
(iv) 7% sucrose, and (v) 0.02% PS80.
and has a pH of 6.
Embodiment 55. 42. The method of any one of embodiments 1-41, wherein the subject is HBeAg negative or HBeAg positive.

Below, specific examples are provided that illustrate embodiments and aspects of the present disclosure. However, this disclosure should not be limited in scope by the particular embodiments described herein. The following preparations and examples are provided to enable those skilled in the art to more clearly understand and practice this disclosure. However, the present disclosure is not limited in scope by the illustrated embodiments. Indeed, various modifications of the disclosure, in addition to those described herein, will become readily apparent to those skilled in the art from the foregoing description, accompanying drawings, and the following examples. All such modifications are within the scope of the following claims.
[Example 1]

Generation and Testing of Modified Antibodies Analysis of several HBC34 antibody variants from PCT publication no. ) revealed the cysteine amino acid. Without wishing to be bound by theory, it is possible that unpaired cysteine residues are potentially reactive and can trigger aggregation through intramolecular scrambling or intermolecular disulfide formation. The mutant form of HBC34-V7 (WO 2017/060504) was modified to replace the cysteine amino acid at position 40 with serine (thereby generating “HBC34-V34”) or alanine (thereby generating “HBC34-V35”) . The nucleotide sequences encoding these additional variant antibodies were codon-optimized and the antibodies were expressed as IgG1 (g1m17, 1 allotype) in ExpiCHO™ cells (ThermoFisher). The codon-optimized nucleotide sequences encoding the VH and VL domains of HBC34-V35 are shown in SEQ ID NOs: 103 and 104, respectively.

The ability of HBC34-V34 and HBC34-V35 to bind antigen was investigated using a direct antigen binding ELISA. HBC34-V7 was used as a comparator. As shown in Figure 1, both HBC34-V34 and HBC34-V35 efficiently bind two recombinant HBsAg antigens (“adw”, upper panel; “adr”, lower panel), and HBC34-V35 , the binding was very similar to that of the parent HBC34-V7.

Mutant antibodies were tested for binding to all known HBsAg genotypes [(A)-(J)]. Briefly, human epithelial cells (Hep2 cells) were transfected with plasmids expressing each of the 10 HBV genotypes A, B, C, D, E, F, G, H, I, and J HBsAg. did. All antibodies were tested at multiple concentrations for staining of transiently transfected permeabilized cells. Two days after transfection, Hep2 cells were harvested, fixed, and permeabilized with saponin for immunostaining with HBC34 and five selected mutants. HBC34-V7 was included as a comparator. Antibody binding to transfected cells was analyzed using a Becton Dickinson FACSCanto2 (BD Biosciences) with FlowJo software (TreeStar). As shown in Figures 2A-2J, HBC34-V34 and HBC34-V35 recognized all 10 HBV HBsAg genotypes. HBC34-V35 showed slightly stronger staining than HBC34-V34.

These data indicate that antibody variants HBC34-V34 and HBC34-V35 broadly recognize HBsAG and bind to HBsAG at levels comparable to HBC34-V7.
[Example 2]

HBC Antibodies with Modified Fc Regions Bind Antigen Efficiently Modifications of the Fc region can provide benefits to therapeutic antibodies. HBC34-V35 was expressed as an IgG1 containing a wild-type Fc, or containing an Fc containing the "MLNS" mutation (M428L/N434S), or containing MLNS in combination with "GAALIE" (G239A/A330L/I332E). Ta. Each construct was tested for binding to recombinant HBsAg (adw) in two separate antigen binding ELISA experiments. Three (3) lots of HBC34-v35 (wild type Fc) were tested. Two (2) lots of HBC34-V35-MLNS and two (2) lots of HBC34-V35-MLNS-GAALIE were tested. HBC34v7 (one lot) was tested as a comparator.

As shown in Figures 3A and 3B, the introduced Fc mutations did not affect the antigen binding activity of HBC34-V35. EC50 values were generally low, although there was some variation between the various constructs and between the two experiments.
[Example 3]

Additional Functional Tests In vitro and in vivo neutralization tests are performed using HBC34-V35, HBC34-V35-MLNS, and HBC34-V35-MLNS-GAALIE. In one study, antibodies were tested for neutralizing activity using HBV-infected mouse PXB cells. In another study, antibodies were tested using human hepatocytes infected with genotype C HBV.

Both tests use hebusbulin (human hepatitis B immunoglobulin) as a positive control. The following data is captured at multiple time points: HBV DNA quantification; HBsAg quantification; HBeAg quantification; and hAlb quantification.
[Example 4]

Identification and Characterization of Human Monoclonal Antibody HBC24 Human monoclonal antibodies were isolated from human patients in a manner similar to that described in Traggiai E. et al., 2004, Nat Med 10(8): 871-5. . The antibody was characterized by determining the nucleotide and amino acid sequences of its variable regions and complementarity determining regions (CDRs) therein and was named "HBC24." Therefore, HBC24 is an IgG1 type fully human monoclonal antibody with CDR, V _H and V _L sequences as shown in Table 3 above. Exemplary nucleotide sequences encoding the V _H and V _L of HBC24 are shown in Table 4.
[Example 5]

HB Antigen Clearance and Viral Entry Inhibition in a Mouse Model Immunodeficient mice transplanted with human hepatocytes were used to test the effectiveness of the anti-HBV antibodies of the present disclosure in eliminating HBsAg. Briefly, primary human hepatocytes were transplanted into SCID mice in which mouse hepatocytes had been previously disrupted by enzymes. The mice were T cell and B cell deficient mice. This model is useful for studying HBV infection, including entry, spread, cccDNA regulation, hepatocyte endogenous immune responses, and viral integration into the host genome.

On day −28, mice were inoculated with rAAV8-1.3HBV strain ayw, type D by tail vein injection at 1.0×10 ⁷ viral genomes per mouse. Treatment on day 0. AAV/HBV infected mice (n=4 per treatment group) were administered PBS (control) or HBC34-v35 (1, 5, or 15 mg/kg ip, 2×/week). The antibody was made into a mouse by removing the antigen-binding Fab region.

Plasma and serum samples were collected periodically throughout the study to determine viral load, HBV DNA (by PCR), and HB Ag (HBsAg, HBeAg, HBcrAg). Mice were sacrificed at 6 weeks. As shown in Figures 4-7, treatment with the highest dose of HBC34-v35 reduced viral load and viral entry into hepatocytes.
[Example 6]

Generation and Functional Testing of Germlined Variants of HBC24 HBC24 is analyzed relative to germline sequences for the presence of somatic mutations in the variable regions. Identified somatic mutations are reverted to germline sequences to generate HBC24 variants. HBC24 and variants are tested for binding (in vitro) and neutralization (in vitro; in vivo) of HBV and HBD serotypes using the assays described in Examples 1 and 3.
[Example 7]

Introduction of Fc modifications to HBC24 and mutants Additional HBC24 mutants are generated containing the MLNS and GAALIE mutations in both Fc monomers. The HC amino acid sequences of selected variants are shown in SEQ ID NOs: 120 and 121. The variants were tested for (1) in vitro binding to antigen; (2) in vitro neutralization of HBV serotypes using the assays described in Examples 1 and 3.
[Example 8]

In Vitro Effector Function Tests The Fc-modified HBC34 antibody has (1) the ability to bind and complement human FcγR; (2) the ability to activate FcγRIIa, FcγRIIb, and FcγRIIIa; and (3) promote ADCC. , conducted an in vitro study to examine its ability to activate human natural killer (NK) cells. The test substances, cell lines, and reagents used were as listed in Tables 5-7 below. The following abbreviations are used in this example: GLP = Good Laboratory Practice; ADCC = Antibody Dependent Cytotoxicity; ADCP = Antibody Dependent Cell Phagocytosis; Fc = Crystalline Fragment; HBsAg = Hepatitis B Surface Antigen. mAb = monoclonal antibody; PBS = phosphate buffered saline; UHPL-SEC = ultra-high performance liquid size exclusion chromatography; ATCC = American Type Culture Collection; FcγR = Fc gamma receptor; CHO cells = Chinese hamster ovary cells; RLU = Relative luminescence; BLI = biolayer interferometry.

Table 5. Test substances

Table 6. Cell lines

Table 7. Other reagents

Experimental Procedures Measurement of Binding to Human Fcγ Receptors The binding of HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE to human FcγRs was measured with an Octet instrument (BLI, Biolayer Interferometry). Briefly, 2 μg/ml of His-tagged human FcγRs (FcγRIIa allele H131, FcγRIIa allele R131, FcγRIIAa allele F158, FcγRIIIa allele V158 and FcγRIIb) was captured with an anti-penta-His sensor for 6 minutes. The FcγR-loaded sensor was then injected with 2 μg/ml of each mAb in the presence of 1 μg/ml of affiniPure F(ab') _2- fragment goat anti-human IgG, F(ab') ₂ -fragment specific (cross-linking human mAbs through Fab fragments). ml of kinetics buffer solution (pH 7.1) for 4 minutes, followed by a dissociation step in the same buffer for an additional 4 minutes (right part of the plot). Binding and dissociation profiles were measured in real time as changes in interference patterns using Octet RED96 (ForteBio).

Measurement of binding to human complement protein C1q The binding of HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE to human complement was measured with an Octet instrument (BLI, Biolayer Interferometry). Briefly, an anti-human Fab (CH1-specific) sensor was used to capture intact IgG1 of HBC34v35 MLNS and HBC34-V35-MLNS-GAALIE mAbs at 10 μg/ml for 10 min through the Fab fragments. The IgG-loaded sensor was then exposed to a kinetics buffer solution (pH 7.1) containing 3 μg/ml of purified human C1q for 4 min (left part of the plot), followed by a dissociation step in the same buffer for an additional 4 min. (right part of plot). Binding and dissociation profiles were measured in real time as changes in interference patterns using Octet RED96 (ForteBio).

Preparation of human NK cells from whole blood NK cells were freshly isolated from EDTA whole blood using the MACSxpress® NK isolation kit according to the manufacturer's instructions. Briefly, anticoagulated blood was mixed with 15 ml of NK isolation cocktail in a 50 ml tube and incubated for 5 minutes at room temperature using a rotator at approximately 12 revolutions per minute. The tube was then placed in the magnetic field of the MACSxpress® Separator for 15 minutes. Magnetically labeled cells adhere to the walls of the tube, while aggregated red blood cells settle to the bottom. Target NK cells were then collected from the supernatant while the tube was still inside the MACSxpress® Separator. NK cells were centrifuged, treated with distilled water to remove residual red blood cells, centrifuged again, and finally resuspended in AIM-V medium.

Determination of antibody-dependent NK cell killing MAbs were serially diluted 10-fold from 100 μg/ml to 0.001 μg/ml in AIM-V medium. Target cells (PLC/PRF/5; MacNab, et al., British Journal of Cancer, 34(5), 1976) were added to round-bottom 384-well plates at 7.5 x ¹⁰ cells/well in 23 μl, then Serially diluted antibodies were added to each well (23 μl per well) and the antibody/cell mixture was incubated for 10 minutes at room temperature. After incubation, human NK cells were added at a cell density of 7.5×10 ⁴ /well in 23 μl, giving an effector to target ratio of 10:1. Control wells used to measure maximum lysis (containing target cells with 23 μl of 3% Triton x-100) and spontaneous lysis (containing target and effector cells without antibody) were also included. Plates were incubated for 4 hours at 37°C with 5% _CO2 . Cell death was determined by measuring lactate dehydrogenase (LDH) release using an LDH detection kit according to the manufacturer's instructions. Briefly, plates were centrifuged for 4 minutes at 400×g and 35 μl of supernatant was transferred to a flat bottom 384-well plate. LDH reagent was prepared and 35 μl was added to each well. Absorbance at 490 nm and 650 nm was measured once every 2 minutes for 8 minutes using a kinetic protocol. Percent specific lysis was determined by applying the following formula: (specific release - spontaneous release)/(maximum release - spontaneous release) x 100.

Determination of Antibody-Dependent NK Cell Activation Activation of primary NK cells was determined using fresh cells from two donors previously genotyped for homozygous high (V158 allele) or low (F158 allele) affinity FcγRIIIa expression. tested using cells isolated in Serial dilutions of mAb (10-fold serially diluted from 100 μg/ml to 0.0001 μg/ml in AIM-V medium) were incubated with NK cells for 4 hours. NK cell activation was measured by flow cytometry by staining NK cells with anti-CD107a mAb (anti-CD107 PE, BioLegend, using a 1/35 dilution) as a functional marker of NK cell activity.

Determination of antibody-dependent activation of human FcγRIIIa HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE were serially diluted 4-fold from 5 μg/ml to 0.076 μg/ml in ADCC assay buffer. Target antigen (Engerix B, HBsAg from Glaxo SmithKline) was added to a white flat bottom 96-well plate at 0.6 μg/ml in 25 μl, then serially diluted antibody was added to each well (25 μl per well) and antibody/cell The mixture was incubated for 10 minutes at room temperature. Effector cells for ADCC Bioassay were thawed and added at a cell density of 7.5×10 ⁴ /well in 25 μl (final HBsAg concentration was 0.2 μg/ml). Control wells used to measure antibody-independent activation (containing HBsAg and effector cells but no antibody) and spontaneous luminescence of the plate (wells containing only ADCP assay buffer) were also included. Ta. Plates were incubated for 24 hours at 37°C with 5% _CO2 . Activation of human FcγRIIIa (V158 or F158 mutant) in this bioassay results in NFAT-mediated expression of the luciferase reporter gene. Luminescence was measured in a luminometer using the Bio-Glo-™ Luciferase Assay Reagent according to the manufacturer's instructions. Data (ie, specific FcγRIIIa activation) are expressed as the average relative light unit (RLU) compared to background applying the following formula: (RLU at mAb concentration x - RLU of background).

Determination of antibody-dependent activation of human FcγRIIa HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE were serially diluted 5-fold from 50 μg/ml to 0.00013 μ/ml in ADCP assay buffer. Target antigen (HBsAg from Engerix B) was added at 0.6 or 6 μg/ml in 25 μl to a white flat bottom 96-well plate, then serially diluted antibody was added to each well (25 μl per well) and antigen/antibody was added to each well (25 μl per well). Incubated for 25 minutes at room temperature. Effector cells for the FcγRIIa activation bioassay were thawed and added at a cell density of 50.0×10 ⁴ /well in 25 μl (final HBsAg concentration was 0.2 or 2 μg/ml, respectively). Control wells used to measure antibody-independent activation (containing HBsAg and effector cells but no antibody) and spontaneous luminescence of the plate (wells containing only ADCP assay buffer) were also included. Ta. Plates were incubated for 23 hours at 37°C with 5% _CO2 . Activation of human FcγRIIa (H131 variant) in this bioassay results in NFAT-mediated expression of the luciferase reporter gene. Luminescence was measured in a luminometer using the Bio-Glo-™ Luciferase Assay Reagent according to the manufacturer's instructions. Data (i.e., specific FcγRIIa activation) are expressed as the average of relative luminescence (RLU) compared to background applying the following formula: (RLU at mAb concentration [x] - RLU of background ).

Determination of antibody-dependent activation of human FcγRIIb HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE were serially diluted 5-fold from 100 μg/ml to 0.00026 μg/ml in ADCP assay buffer. Target antigen (HBsAg from Engerix B) was added to a white flat-bottomed 96-well plate at 3 μg/ml in 25 μl, then serially diluted antibodies were added to each well (25 μl per well) and the antigen/antibody was incubated for 15 minutes at room temperature. Incubated. Effector cells for the FcγRIIb activation bioassay were thawed and added at a cell density of 75.0×10 ⁴ /well in 25 μl (final HBsAg concentration was 1 μg/ml). Control wells used to measure antibody-independent activation (containing HBsAg and effector cells but no antibody) and spontaneous luminescence of the plate (wells containing only ADCP assay buffer) were also included. Ta. Plates were incubated for 20 hours at 37°C with 5% _CO2 . Activation of human FcγRIIb in this bioassay results in NFAT-mediated expression of the luciferase reporter gene. Luminescence was measured in a luminometer using the Bio-Glo-™ Luciferase Assay Reagent according to the manufacturer's instructions. Data (i.e., specific FcγRIIb activation) are expressed as the average of relative luminescence (RLU) compared to background applying the following formula: (RLU at mAb concentration [x] - RLU of background ).

Determination of antibody binding to the human hepatocellular carcinoma cell line PLC/PRF/5 PLC/PRF/5 cells were trypsinized for 5 min at 37°C, transferred to 7 ml growth medium, and centrifuged at 400 x g for 4 min at 4°C. Separated and washed extensively in PBS at 4°C. Some cells were fixed with 4% formaldehyde (20 min at 4°C); other cells were fixed and then permeabilized with permeabilization buffer (20 min at 4°C). Cell pellets were resuspended in 2.64 ml of wash buffer (fixed cells) or permeabilization buffer (fixed & permeabilized cells) (Table 7) and dispensed at 200 μl/well into 96-well round bottom plates (100 μl/well). '000 cells/well). The plate was centrifuged at 400g for 4 minutes at 4°C. Serial 1:5 5-point dilutions of the test antibody starting at a final concentration of 10 μg/ml were added to the cell-containing wells and incubated on ice for 30 minutes. After two washes at 4°C, 400 x g, 4 min with wash buffer (fixed cells) or permeabilization buffer (fixed & permeabilized cells), 50 μl/well of Alexa Fluor® 647-conjugated secondary antibody (Table 7) was added to the cells and incubated on ice for 20 minutes. Cells were washed two more times with wash buffer (fixed cells) or permeabilization buffer (fixed & permeabilized cells) and 200 μl/well of wash buffer (fixed cells) or permeabilization buffer (fixed & permeabilized cells). treated cells) and the signal (MFI, mean fluorescence intensity) was quantified using a cytofluorometer (BD FACSCanto II).

Results Direct antiviral mechanisms are important for neutralizing HBV in vivo. Indirect Fc-dependent mechanisms of action mediated by the interaction of the Fc region with Fc gamma receptors (FcγRs) on immune cells also have important contributions to the efficiency and mediation of endogenous immune responses in vivo. obtain. FcγR-dependent mechanisms can be assessed in vitro by measuring binding to FcγRs and in antibody-dependent activation of human FcγRs (Hsieh, Y.-T., et al., Journal of Immunological Methods , 441(C), 56-66. doi.org/10.1016/j.jim.2016.12.002).

In this study, HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE were evaluated using biolayer interferometry for their ability to bind to the full set of human FcγRs (FcγRIIIa V158 and F158 alleles, FcγRIIa H131 and R131 alleles, and FcγRIIb). (BLI Octet System, ForteBio) was used for control comparison. As shown in Figures 8A-8E, Fcs with MLNS-GAALIE mutations have altered interaction with FcγR; specifically, Fcs with these mutations have impaired binding to FcγRIIIa and FcγRIIa. binding to FcγRIIb is decreased. Not surprisingly, the binding of HBC34-V35-MLNS-GAALIE to C1q was abolished as measured by biolayer interferometry (Figure 9).

HBC34-V35-MLNS and HBC34-V35-MLNS-GAALIE were also tested for their ability to activate human FcγRIIIa and FcγRIIa using a cell-based reporter bioassay. These assays utilize Jurkat cells modified with an NFAT-mediated luciferase reporter to reflect activation of human FcγRs. HBC34v35-MLNS poorly or did not activate human FcγRIIIa and FcγRIIa in the presence of HBsAg, whereas HBC34-V35-MLNS-GAALIE induced dose-dependent activation of all FcγRs tested. (FIGS. 10A, 10B, 11A, and 11B). In contrast, HBC34-V35-MLNS-GAALIE did not activate FcγRIIb even when tested at 100 μg/ml (Figure 12).

Natural killer cells (NK) isolated from human peripheral blood mononuclear cells of one donor previously genotyped for expression of heterozygous high (V158) and low (F158) affinity FcγRIIIa (F/V). was used to also measure ADCC activity. Using isolated NK cells, HBC34v35; HBC34v35-MLNS; HBC34-V35-MLNS-GAALIE; or another mAb (17.1.41, targeting another epitope on the antigenic loop of HBsAg; Galun, E., et al., Hepatology, doi.org/10.1053/jhep.2002.31867). Killing of hepatocellular carcinoma cell line PLC/PR/5 was measured. No killing was observed in the presence of HBsAg-specific mAbs HBC34v35, HBC34v35-MLNS, HBC34-V35-MLNS-GAALIE and 17.1.41 (Figure 13A). The observed lack of antibody-dependent killing of PLC/PR/5 cells may be related to poor expression of HBsAg on the surface of these cells (Figure 13B) and wish to be bound by theory. However, it may not be sufficient to trigger death by NK cells. Conversely, high levels of HBsAg were detected on HBC34v35 and 17.1.41 when PLC/PR/5 cells were fixed and permeabilized. This indicates that most of the HBsAg is found either intracellularly or in secreted form (ie, subviral particles) (Figure 13B).

Activation of primary human NK cells (V/F) in the presence of HBC34v35-MLNS or HBC34-V35-MLNS-GAALIE and HBsAg was also examined using anti-CD107a mAb. The data are shown in Figures 14A and 14B.

These in vitro data demonstrate that HBV-specific binding proteins of the present disclosure with GAALIE Fc mutations bind and activate low-affinity activating FcγRIIa and FcγRIIIa more effectively than non-GAALIE Fc parent antibodies. ing. GAALIE-containing binding proteins also did not bind or activate low affinity inhibitory FcγRIIb. GAALIE-containing binding proteins also do not bind C1q. Moreover, GAALIE-containing binding proteins do not promote ADCC in hepatocellular carcinoma cells, but activate human NK cells in the presence of soluble HBsAg.
[Example 9]

Phase 1 clinical trial of HBC34-v35-MLNS-GAALIE A multi-center, phase 1, randomized, placebo-controlled study was conducted to develop HBC34-v35-MLNS-GAALIE (the heavy chain amino acid sequence shown in SEQ ID NO: 91). and the light chain amino acid sequence shown in SEQ ID NO: 93). The study sites are: Part A (single center) and Part B/C (multicenter).

In Part A (up to 40 participants), the primary objective is to evaluate the safety and tolerability of HBC34-v35-MLNS-GAALIE in healthy adult subjects. Secondary objectives were to characterize the serum pharmacokinetics (PK) of HBC34-v35-MLNS-GAALIE in healthy adult subjects and to determine the immunogenicity [anti-drug antibodies ( ADA) induction].

In Parts B (up to 56 participants) and C (up to 24 participants), the primary objective is to evaluate the safety and tolerability of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis. It is. Secondary objectives were to characterize the serum PK of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis; and to evaluate the immunogenicity (induction of ADA) of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis. Exploratory objectives include: To assess the effects of HBC34-v35-MLNS-GAALIE on additional viral parameters; HBC34-v35 on immune responses (or exploratory biomarkers) in adult subjects with chronic HBV infection without cirrhosis; - To evaluate the efficacy of MLNS-GAALIE; and to determine the impact of host genetic polymorphisms (or exploratory biomarkers) on the response to HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis. One example is to evaluate.

Evaluation Criteria Details For Part A, the primary endpoints of this study are:
・Clinical evaluation including, but not limited to, the incidence of treatment-emergent adverse events (TEAEs) and laboratory test results. Secondary endpoints of this study are as follows:
- HBC34-v35-MLNS-GAALIE serum-free PK parameters, such as: C _max , Clast, T _max , T _last , AUC _inf , AUC _last , %AUC _exp , t _1/2 , λ _z , V _z (IV only) , CL (IV only), V _z /F (SC only), and CL/F (SC only)
Incidence and titer of ADA against HBC34v-35-MLNS-GAALIE (if applicable)
For Parts B/C, the primary endpoints of this study are:
Clinical evaluation including, but not limited to, incidence of TEAEs and laboratory test results Secondary endpoints for this study are:
HBC34-v35-MLNS-GAALIE serum-free and complete PK parameters, such as: C _max , C _last , T _max , T _last , AUC _inf , AUC _last , %AUC _exp , t _1/2 , λz, V _z /F , and CL/F.
Incidence and titer of ADA against HBC34-v35-MLNS-GAALIE (if applicable)
Maximum reduction in serum HBsAg from baseline (day 1 pre-dose) Exploratory endpoints for this study include:
Evaluation of additional viral parameters (e.g.: HBV RNA and HBcrAg)
- Analysis of host immune responses - Analysis of host factors as determined by RNA sequencing - Fc gamma receptor (FcγR) polymorphisms as determined by genotyping - IgG allotypes as determined by genotyping.

Expected number of subjects Part A: Up to 40 healthy adult subjects Part B: HBeAg negative, HBsAg <1000 IU/mL, on nucleoside reverse transcriptase inhibitor (NRTI) therapy, without cirrhosis Up to 56 adult subjects with chronic HBV infection Part C: Up to 24 adult subjects with chronic HBV infection without cirrhosis on NRTI therapy with HBsAg ≧1000 IU/mL

Diagnosis and Key Inclusion Criteria Inclusion criteria for Part A include:
Healthy adult subjects aged 18 years (or legal age of consent, whichever is older) weighing ≧40 kg to ≦125 kg. Patients must be in good health as determined by medical history (e.g., chronic conditions such as hypertension, hyperlipidemia, gastroesophageal reflux disease, asthma, anxiety, and depression must be well controlled) and in good physical condition. There are no clinically significant findings from laboratory tests, 12-lead ECG, vital signs, and laboratory values. Female subjects must have a negative pregnancy test or confirmation of postmenopausal status. Postmenopausal status is defined as 12 months of amenorrhea without another medical cause. Women of Childbearing Potential (WOCBP) must have a negative blood pregnancy test at screening and a negative urine pregnancy test on Day 1, cannot be breastfeeding, and must not be present in the study for 14 days prior to study drug administration. Must be willing to use a highly effective method of contraception as disclosed herein for up to 40 weeks after drug administration.

Male subjects with a fertile female partner must agree to meet one of the following contraceptive requirements from the time of study drug administration until 40 weeks after study drug administration: Submission of documentation of azoospermia or vasectomy with male condom use plus frequent use of effective contraception by the partner. Male subjects must also agree not to donate sperm from the time of study drug administration until 40 weeks after study drug administration. Patients agree not to donate blood during the continuation of the study.

Patients are willing to comply with study requirements and are able to provide written informed consent.

Part B/C inclusion criteria include:
1. 18 years old (or legal age of consent, whichever is older) - 65 years old 2. Weight ≥40 kg to ≤125 kg, chronic HBV infection [defined by two positive serum HBsAg, HBV DNA, or HBeAg tests at least 6 months apart based on previous or current laboratory evidence (6 months apart)] Any combination of these tests performed is acceptable).
3. 4. No liver cirrhosis. Be on NRTI therapy for at least 2 months at screening and HBeAg negative. Examples of NRTI therapies include, but are not limited to, tenofovir disoproxil/tenofovir alafenamide; entecavir; lamivudine; adefovir/adefovir dipivoxil.
5. HBV DNA <100 IU/mL at screening
6. HBsAg>lower limit of detection7. HBsAg < 3000 IU/mL at screening (Part B only); Cohort 1b (6 mg SC) enrolled participants with HBsAg < 1000 IU/mL 8. HBsAg≧1000IU/mL at screening (Part C only)
9. HBeAg negative at screening (Part B only)
10. Negative anti-HBs during screening
11. Must be in good health as determined by medical history, excluding chronic HBV infection (e.g., chronic conditions such as hypertension, hyperlipidemia, gastroesophageal reflux disease, asthma, anxiety, and depression must be well controlled) There should be no clinically significant findings from physical examination, 12-lead ECG, vital signs, and laboratory values.
12. Female subjects must have a negative pregnancy test or confirmation of postmenopausal status. Postmenopausal status is defined as 12 months of amenorrhea without another medical cause. Women of childbearing potential must have a negative blood pregnancy test at screening and a negative urine pregnancy test on Day 1, must not be breastfeeding, and must have a negative blood pregnancy test at screening and a negative urine pregnancy test on day 1, and must not be breastfeeding and have no symptoms starting 14 days before study drug administration. Must be willing to use an effective method of contraception until 40 weeks.
13. Male subjects with a fertile female partner must agree to meet one of the following contraceptive requirements from the time of study drug administration until 40 weeks after study drug administration: Submission of documentation of azoospermia vasectomy with or use of one of the contraceptive options listed for male condom use plus partner WOCBP contraception (see herein). Male subjects must also agree not to donate sperm from the time of first study drug administration until 40 weeks after study drug administration.
14. Willing to comply with study requirements and able to provide written informed consent.

Fertility control methods that are considered to be highly effective include:
Use of established combination oral, vaginal, or transdermal hormonal contraceptive methods (containing estrogen and progesterone) that inhibit ovulation, or use of oral, injectable, or implantable hormonal contraceptive methods that inhibit ovulation. Use of established progesterone only. It is currently unknown whether HBC34-v35-MLNS_GAALIE affects the effectiveness of hormonal contraceptive methods; therefore, additional forms of contraception (i.e., barrier methods) were used throughout the study and for 40 weeks after study drug administration. Recommended for use.
・Insertion of an intrauterine contraceptive device ・Insertion of an intrauterine hormone-releasing system ・Contraception of the male partner (by submitting proper post-vasectomized evidence of the absence of sperm in the semen; The male partner who underwent ductal resection should be the subject's only partner)
- True abstinence from contact with the opposite sex, if in line with the subject's preferred and normal life. Cyclic abstinence (eg, calendars, ovulation methods, symptomatic temperature methods, postovulatory methods) and extravaginal ejaculation are not acceptable methods of contraception. Abstinent subjects should use one of the contraceptive methods listed above if they begin sexual relations during the study and up to 40 weeks after study drug administration, or while the subject is being followed in the study, whichever is longer. must agree.
Barrier methods in conjunction with hormonal contraception as described above Postmenopausal status is defined as 12 months of amenorrhea without another medical cause.
Male subjects with a female partner of childbearing potential must agree to meet one of the following contraceptive requirements from the time of study treatment administration until 40 weeks after study drug administration:
- Vasectomy with documentation of azoospermia - Use of one of the contraceptive options listed above (hormonal contraception, intrauterine device) for male condom use plus partner WOCBP contraception. Participants must also agree not to donate sperm for 40 weeks after the last dose of study drug.

Study Participation Period Part A: The study drug treatment period is a single dose. Estimated total study time, including screening and follow-up for each subject, is up to 28 weeks.
Part B/C: Study drug treatment period is a single dose. Estimated total study time, including screening and follow-up for each subject, is up to 44 weeks.

Follow-up Period Part A: All subjects will be followed for 24 weeks after study drug administration.
Part B/C: All subjects will be followed for 8 weeks after study drug administration. Subjects with >2-fold HBsAg reduction at week 8 will receive a total of up to 40 weeks or until HBsAg reduction is <2-fold compared to baseline at 2 consecutive sampling points, whichever comes first. Undergo extended follow-up. Extended follow-up may be discontinued based on emerging data.

Study Design The Safety Review Committee (SRC) will evaluate the continued safety, tolerability, and antiviral activity data (parts B and C only) at specified time points based on available data collected throughout the study. Conduct a review. The primary data reviewed by the SRC for dose escalation and enrollment of any cohort will be enumerated throughout the protocol, but additional relevant data from other cohorts will also be reviewed by the SRC as indicated to make decisions. Inform.
The trial will be conducted in three parts:
Part A: A randomized, double-blind, placebo-controlled, single-dose dose escalation study of HBC34-v35-MLNS-GAALIE administered to healthy adult subjects by subcutaneous (SC) or intravenous (IV) infusion. SAD) test.
Part B: Randomization of HBC34-v35-MLNS-GAALIE administered by SC injection to adult subjects with chronic HBV infection without cirrhosis who are HBeAg negative and HBsAg <1000 IU/mL on NRTI therapy. A double-blind, placebo-controlled, SAD study.
Part C: Randomized, double-sided study of HBC34v35-MLNS-GAALIE administered by SC injection to adult subjects with chronic HBV infection without cirrhosis who are on NRTI therapy and have HBsAg ≧1000 IU/mL. Blinded, placebo-controlled, SAD study.

Overall Risk/Benefit Assessment The potential risks to healthy adult subjects are not specific to HBC34-v35-MLNS-GAALIE, based on the general safety risks observed with mAb class therapies: anaphylaxis and others. Serious allergic reactions and injection/infusion related reactions. In particular, the risk of developing such a condition following administration of HBC34v35-MLNS-GAALIE is unknown.
Part A of the trial will collect information regarding the safety and tolerability of HBC34v35-MLNS-GAALIE, as well as relevant data regarding PK profile and anti-drug antibody (ADA) generation. HBC34-v35-MLNS-GAALIE is not expected to provide benefit to healthy subjects enrolled in Part A of this study. Subjects will be monitored for important potential risks and routine pharmacovigilance and risk minimization activities will be conducted.
The potential benefits of HBC34-v35-MLNS-GAALIE in subjects with chronic HBV infection compared to current standard treatments are as follows:
- Reduction of serum HBsAg, inhibition of intrahepatic spread of HBV, elimination of infected hepatocytes, and stimulation of adaptive immune responses against HBV - Pan-genotypic therapy for HBV infection that is well tolerated and administered SC for a finite period of time In addition to anaphylaxis, other serious allergic reactions and injection/infusion-related reactions, potential risks associated with administration of HBC34-v35-MLNS-GAALIE to subjects with chronic HBV infection include immune complexes. This includes hepatotoxicity due to elimination of infected hepatocytes via cytotoxic T cells induced by disease and ADCC/ADCP and/or vaccine effects. The Part B/C study design includes several elements that reduce these risks:
- Part B enrolls subjects with serum HBsAg <1000 IU/mL to reduce the risk of immune complex disease and hepatotoxicity. Additionally, Part B safety data will be reviewed by the SRC prior to enrolling subjects with potentially higher baseline HBsAg values into the appropriate Part C of the trial.
- Parts B and C are in NRTI, have HBV DNA <100 IU/mL at screening, and have the following characteristics at baseline: ALT or AST ≤2 x ULN, no history of hepatic decompensation, and significant Subjects with good liver reserve and low levels of liver inflammation, as determined by the absence of fibrosis and cirrhosis, will be enrolled.
- Two sentinel subjects will be randomized 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo. These sentinel subjects will be monitored for at least 72 hours after dosing, and if the investigator(s) do not have safety concerns, the remaining 6 subjects in the same cohort will be dosed (5 active and 5 placebo). 1 person).
4 after dose administration to illustrate the expected timing of hepatotoxicity due to potential immune complex disease and elimination of infected hepatocytes via cytotoxic T cells induced by ADCC/ADCP and/or vaccine effects. Dose escalation will occur after SRC review of up to 1 week of available safety data.
- Design safety monitoring to include liver function tests, urinalysis, renal function, vital signs, and physical examination findings to detect evidence of HBC34-v35-MLNS-GAALIE-related immune adverse events.

Part A
Three consecutive cohorts in Part A evaluate 90 mg, up to 300 mg, and up to 900 mg administered by SC injection. The SRC will review available clinical and laboratory safety data for all available subjects in the cohort prior to dose escalation, up to two weeks post-dose. Two optional cohorts from Part A may be added evaluating up to 900 mg and 3000 mg administered by IV infusion. Enrollment of any of these cohorts can occur after SRC review of available Week 2 data from all available subjects in Cohort 3a (up to 900 mg SC).
All SC cohorts in Part A (cohorts 1a, 2a, and 3a) are enrolled sequentially, but additional cohort(s) are expected to have an acceptable safety and tolerability profile in the preceding cohorts of Part A. Cohorts may be enrolled simultaneously if a dose level that is at or below a dose level previously found to have .
In each cohort, two sentinel subjects will be randomized 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo. These subjects will be dosed and monitored for at least 24 hours in an inpatient setting; if the investigator does not have safety concerns, the remaining subjects in the same cohort will be dosed. The remaining subjects will be randomized 5:1 to receive HBC34-v35-MLNS-GAALIE or placebo.
The maximum dose escalation factor for Part A will not exceed 5 times.

Part B
The first cohort of Part B (Cohort 1b) will be enrolled after SRC review of the available Week 2 data from all available subjects in Cohort 1a (90 mg SC).

For Part B, five cohorts evaluating 6 mg (cohort 1b), 18 mg (cohort 2b), up to 75 mg (cohort 3b), up to 300 mg (cohort 4b), and up to 900 mg (cohort 5b) administered by SC injection plan. The SRC will review available clinical and laboratory safety data and antiviral activity data for up to 4 weeks post-dose on all available subjects in the preceding cohort prior to dose escalation.
Part B may add two optional cohorts that follow the same dosing schedule. Any cohort may be dosed at a lower, equivalent, or intermediate dose level compared to the dose level studied in the planned Part B cohort, or at a dose level not exceeding 900 mg in Cohort 5b. It may be administered after. The maximum dose level for any cohort in Part B will not exceed the highest single dose found to have an acceptable safety and tolerability profile in Part A. Any cohort may be enrolled at any time within the Part B planned cohort upon SRC approval.

All Part B cohorts should be enrolled sequentially, but additional cohort(s) should have acceptable safety and tolerability profiles in Part A and Part B prior cohorts. Cohorts may be enrolled simultaneously if a pre-discovered dose level or a lower dose level is being investigated.

In each cohort, two sentinel subjects will be randomized 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo by SC injection. These subjects will be dosed and monitored for at least 72 hours after dosing (including in-hospital monitoring for at least the first 24 hours); unless the investigator(s) have safety concerns, the rest of the same cohort will be administered to subjects. The remaining subjects will be randomized 5:1 to receive HBC34-v35-MLNS-GAALIE or placebo by SC injection.

The maximum dose escalation factor for Part B will not exceed 5 times.

Part C
Part C is optional and may be implemented based on the acceptable safety and tolerability profile of HBC34-v35-MLNS-GAALIE in HBeAg negative subjects with Part B HBsAg levels <1000 IU/mL. The first Part C cohort will be administered at Week 4 for all Part A and Part B subjects for the Part B subject cohort receiving commensurate or higher doses compared to the Part C proposed starting dose level. It will be registered after the SRC reviews the available data up to the hospital visit.

Three optional cohorts may be enrolled in Part C. Each cohort may evaluate up to 900 mg administered by SC injection, and the dose utilized in the Part C cohort was found to have an acceptable safety and tolerability profile by the SRC in Part B. Do not exceed the highest dose level. Cohorts may be enrolled simultaneously.

In each cohort, two sentinel subjects will be randomized 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo by SC injection. These subjects will be dosed and monitored for at least 72 hours post-dose (including in-hospital monitoring over at least the first 24 hours); unless the investigator(s) have safety concerns, the rest of the same cohort will be administered to subjects. The remaining subjects will be randomized 5:1 to receive HBC34-v35-MLNS-GAALIE or placebo by SC injection.

Test procedure part A
Screening Healthy adult subjects will be enrolled in one of the five cohorts in Part A (3 planned cohorts, 2 arbitrary cohorts). Screening will occur no more than 4 weeks prior to the Day 1 visit and will include written informed consent according to the Schedule of Assessment (SoA), determination of eligibility, collection of demographic and medical history, physical examination, vital signs, and laboratory tests. , 12-lead electrocardiogram (ECG), and other assessments.
- Eligible subjects will be admitted to the study site on Day -1 or Day 1. Evaluate eligibility criteria related to vital signs, pregnancy testing, drug abuse, blood donation, presence of any clinically significant acute condition, and use of prescription, OTC, herbal, or investigational medications on day 1; Ensure eligibility to continue testing. Any changes to medical history will also be evaluated and recorded. Eligible subjects in each cohort will be randomized to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to study drug administration. Subjects will receive a single dose of study drug (HBC34-v35-MLNS-GAALIE or placebo) on Day 1.
- Adverse events (AEs) related to screening activities will be collected from the time of consent and thereafter; any other events occurring during the screening period will be reported as medical history. All serious adverse events (SAEs) will be collected from the time of consent onward.
Serological parameters of the screening virus are as follows: Active infection with HIV, HCV, and HBV Day of administration (day 1)
- Eligible subjects will be randomized to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours before study drug administration on Day 1.
-Eligible subjects will receive a single dose of study drug on Day 1 and any applicable assessments will be conducted.
- At the beginning of each cohort, two sentinel subjects will be randomized 1:1 to HBC34v-35-MLNS-GAALIE or placebo. These subjects will be administered and monitored for at least 24 hours in an inpatient setting. Vital signs, ECG, symptom-based physical examination(s), and AEs will be reviewed by the investigator; if the investigator has no safety concerns, administer to remaining subjects in the same cohort . The remaining subjects in the cohort will be randomized 5:1 to receive a single dose of HBC34-v35-MLNS-GAALIE or placebo. All subjects will be closely monitored after dose administration.
Follow-up Period - Subjects will be discharged from the hospital after completing all study assessments on day 2. All subsequent study visits will be outpatient.
・Subjects will be required to attend the study site until week 24 for in-person evaluation as per SoA, including but not limited to physical examination, vital signs, clinical tests, PK evaluation, and review of AEs and concomitant medications. Return to

Part B/C
Screening Screening will be conducted no more than 4 weeks prior to the Day 1 visit and will include written informed consent per SoA, determination of eligibility, collection of demographic and medical history, physical examination, vital signs, laboratory tests, 12 Includes lead ECG and other evaluations. Adverse events related to screening activities will be collected from the time of consent and thereafter; any other events occurring during the screening period will be reported as medical history. All SAEs will be collected from the time of consent onwards.
Adult subjects with HBeAg-negative chronic HBV infection without cirrhosis and HBsAg <3000 IU/mL on NRTI therapy for ≥2 months were enrolled in 7 cohorts of Part B (5 planned cohorts, 2 [Cohort 1b (6 mg SC) enrolled participants with HBsAg < 1,000 IU/mL]. Subject screening will occur no more than 4 weeks prior to Day 1 visit. Subjects will be admitted to the study site on Day -1 or Day 1. On day 1, assess eligibility criteria related to NRTI adherence, vital signs, pregnancy testing, presence of any clinically significant acute conditions, hepatic decompensation, and use of prescription, OTC, herbal, or investigational medications. to ensure eligibility to continue the study. Any changes to medical history will also be evaluated and recorded. Eligible subjects in each cohort will be randomized to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to Day 1 study drug administration.
- Subjects in Parts B and C will undergo a Fibroscan evaluation to exclude the presence of cirrhosis. This does not need to be done if the subject has had a fibroscan in the 6 months prior to screening or a liver biopsy in the year prior to screening to confirm the absence of Metavir F3 fibrosis or F4 cirrhosis.
- Screening viral serological parameters are: active infection with HIV, HCV, and hepatitis delta virus. Subjects with a positive HCV serology result may undergo an HCV-RT PCR reflex test to determine eligibility.
Chronic HBV infection is determined at the time of screening and is defined as: two positive serum HBsAg, HBV DNA, or HBeAg tests at least 6 months apart based on previous or current laboratory evidence (6 Any combination of these tests performed months apart is acceptable).
Administration date (1st day)
- Eligible subjects will be randomized to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours before study drug administration on Day 1.
-Subjects will be admitted to the study site on day 1.
-Eligible subjects will receive a single dose of study drug on Day 1 and any applicable assessments will be conducted.
- At the beginning of each cohort, two sentinel subjects will be randomized 1:1 to HBC34-v35-MLNS-GAALIE or placebo. These subjects will be dosed and monitored for at least 72 hours after dosing (including in-hospital monitoring for at least the first 24 hours); unless the investigator(s) have safety concerns, the rest of the same cohort will be administered to subjects. Vital signs, symptom-based physical examination(s), and AEs will be reviewed by the Investigator prior to dosing any additional subjects. The remaining subjects in the cohort will be randomized 5:1 to receive a single dose of antibody composition or placebo. All subjects will be closely monitored after dose administration.
Follow-up Period - Subjects will be discharged from the hospital after completing all study assessments on day 2. All subsequent study visits will be outpatient.
・Subjects will be required to undergo SoA evaluation up to week 8, including but not limited to physical examination, vital signs, clinical tests, PK evaluation, efficacy evaluation, and review of AEs and concomitant medications. Return to study site.
Extended Follow-up Period Subjects with >2-fold HBsAg reduction at week 8 will continue to study at any time until week 40, or until HBsAg decrease is <2-fold compared to baseline at two consecutive sampling points. The patient will return to the clinical trial facility at the earliest for evaluation according to the SoA. Extended follow-up may be discontinued based on emerging data.

Product, Dosage, and Method of Administration HBC34v35-MLNS-GAALIE is supplied as a lyophilized solid, reconstituted in Sterile Water for Injection (USP) at a concentration of 150 mg/mL, and administered as an SC injection or IV infusion. Unit doses are based on volume and method of administration. When reconstituted at 150 mg/mL in Sterile Water for Injection, USP, the formulation contains 20 mM histidine, 7% sucrose, 0.02% PS80 at pH 6 when administered. Placebo is a sterile, preservative-free 0.9% saline solution for IV or SC injection.
Cohort 1a: HBC34v35-MLNS-GAALIE, single dose of 90 mg administered by SC injection Cohort 2a: HBC34v35-MLNS-GAALIE, single dose of up to 300 mg administered by SC injection Cohort 3a: HBC34v35-MLNS - GAALIE, single dose up to 900 mg administered by SC injection Cohort 4a (as appropriate): HBC34v35-MLNS - GAALIE, single dose up to 900 mg administered by IV infusion Cohort 5a (as appropriate): HBC34v35-MLNS - GAALIE, single dose of up to 3000 mg administered by IV infusion Cohort 1b: HBC34v35-MLNS-GAALIE, single dose of 6 mg administered by SC injection Cohort 2b: HBC34v35-MLNS-GAALIE, administered by SC injection Cohort 3b: HBC34v35-MLNS-GAALIE, up to 75 mg single dose administered by SC injection Cohort 4b: HBC34v35-MLNS-GAALIE, up to 300 mg single dose administered by SC injection Dosing Cohort 5b: HBC34v35-MLNS-GAALIE, single dose up to 900 mg administered by SC injection Cohort 6b (as appropriate): HBC34v35-MLNS-GAALIE, single dose up to 900 mg administered by SC injection Cohort 7b (as appropriate): HBC34v35-MLNS-GAALIE, single dose up to 900 mg administered by SC injection Cohort 1c (as appropriate): HBC34v35-MLNS-GAALIE, single dose up to 900 mg administered by SC injection Cohort 2c (as appropriate): HBC34v35-MLNS-GAALIE, single dose up to 900 mg administered by SC injection Cohort 3c (as appropriate): HBC34v35-MLNS-GAALIE, single dose up to 900 mg administered by SC injection

IV=静脈内; SC=皮下 Table 8: Part A Dose Escalation Plan

IV=intravenous; SC=subcutaneous

Part B: Single Dose Dose Escalation Study in Subjects with Chronic HBV Infection In Part B, subjects with chronic HBV infection receive a single dose of study drug. The presence of HBsAg, the therapeutic target of HBC34-v35-MLNS-GAALIE in subjects with chronic HBV infection, alters the potential risks of HBC34-v35-MLNS-GAALIE administration. Potential risks include immune complex disease due to the formation of antigen-antibody complexes, and hepatotoxicity due to elimination of infected hepatocytes via ADCC/ADCP and/or the "vaccine effect." To minimize risk to subjects, Part B is in NRTI and has HBV DNA <100 IU/mL at screening, as determined by absence of fibrosis/cirrhosis and ALT <2 x ULN , performed in subjects with good liver reserve and low levels of liver inflammation.
A cohort of five dose levels will be used for Part B. Doses are escalated approximately 3-4 times to a planned maximum dose of 900 mg administered by SC injection:
Two optional cohorts will enroll up to a maximum dose of 900 mg administered by SC injection. Cohort 7b may be enrolled for the purpose of collecting and evaluating immune response samples and liver fine needle aspirates, if available at selected facilities, but not limited to. These dose levels are based on preclinical animal models and translational PK/PD modeling predicting significant HBsAg reduction for doses ranging from 2-15 mg/kg. Details regarding the Part B dose escalation schedule can be found in Table 9.

SC=皮下 Table 9: Part B Dose Escalation Plan

SC=subcutaneous

Optional Part C: Single Dose Dose Escalation Study in Subjects with Chronic HBV Infection Evaluate the safety, tolerability, and antiviral activity of HBC34-v35-MLNS-GAALIE in subjects with baseline HBsAg levels ≥1000 IU/mL After the safety, tolerability, and antiviral activity of HBC34-v35-MLNS-GAALIE has been established in HBeAg-negative subjects with Part B HBsAg <1000 IU/mL, an appropriate Part C is performed. do. Part C consists of three appropriate dose level cohorts, each evaluating doses up to 900 mg administered by SC injection. Table 10. One or more optional cohorts of Part C may be enrolled for the purpose of collecting and evaluating immune response samples and liver fine needle aspirates at selected facilities, if applicable, but not limited to. .

Table 10: Part C Cohort Summary

Reference therapy, dosage and method of administration:
Subjects randomized to placebo will receive a sterile, preservative-free 0.9% saline solution by SC injection (Parts A, B, and C) or IV infusion (Part A only).

Local Tolerability For all study parts, perform local tolerability assessments according to the evaluation schedule (external appearance of subjects receiving study drug by SC injection; injection site(s) marked and positioned for later observation). The injection site(s) should be monitored for pain/tenderness, swelling, redness, purpura, and pruritus.
The timing of local tolerability assessment for Part A is shown in Figures 15A-15C. The timing of local tolerability assessment for Part B/C is shown in Figures 16A-16E.
At the discretion of the Investigator, unscheduled visits will be permitted as needed to follow up on any unresolved local intolerance symptoms.

Screening for Substance Abuse For Parts A, B, and C of the study, urine will be collected for drug abuse screening. The panel includes amphetamines, cocaine, methadone, and opiates.

Pharmacokinetic Evaluation Blood samples will be collected to assess the concentration of HBC34-v35-MLNS-GAALIE. The time points at which samples are taken for HBC34-v35-MLNS-GAALIE PK analysis in Part A of the study are indicated herein. The time points at which samples are taken for HBC34-v35-MLNS-GAALIE PK analysis in Parts B and C of the study are indicated herein.

Pharmacokinetic analysis part A
The free PK parameters of HBC34-v35-MLNS-GAALIE are computed using standard non-compartmental methods. Parameters include, but are not limited to, serum: C _max , C _last , T _max , T _last , AUC _inf , AUC _last , %AUC _exp , t _1/2 , λ _z , V _z (IV only) , CL (IV only), V _z /F (SC only), and CL/F (SC only). Other parameters are calculated as necessary.
Part B/C
Free and complete PK parameters of HBC34-v35-MLNS-GAALIE are computed using standard non-compartmental methods. Parameters include, but are not limited to, serum: C _max , C _last , T _max , T _last , AUC _inf , AUC _last , %AUC _exp , t _1/2 , λ _z , V _z /F, and Includes CL/F. Other parameters are calculated as necessary.
PK/pharmacodynamic analysis will be performed to investigate the exposure-response relationship between PK parameters and selected antiviral variables.

Antiviral Activity Analysis For Parts B and C, selected data related to the antiviral activity of HBC34-v35-MLNS-GAALIE, such as HBsAg, anti-HBs, HBeAg, anti-HBe, HBV RNA, HBcrAg, and HBV DNA levels, were analyzed. , summarized by cohort and study visit with corresponding changes from baseline (n, mean, SD, median, Q1, Q3, min, and max). A summary (number and percentage of subjects) of HBsAg reduction (defined as undetectable HBsAg measured on two consecutive separate occasions at least 2 weeks apart) is presented by cohort and study visit.

Immunogenicity Blood samples will be taken for analysis of the immunogenic response to determine the presence/absence and titer of anti-drug antibodies (ADA), if applicable, according to the time points defined in the evaluation schedule ( 15A-16E). If desired, samples are characterized for neutralizability potential (NAb) of HBC34-v35-MLNS-GAALIE.

Evaluation of Screening Viral Parameters, Antiviral Activity, and Resistance Surveillance During Parts B and C, evaluation of screening viral parameters includes HBsAg, anti-HBs, HBeAg (qualitative), and HBV DNA.
Post-screening assessments of antiviral activity include HBsAg, anti-HBs, HBeAg (qualitative; should be collected only for Part C subjects who are HBeAg qualitative positive at screening), HBeAg (quantitative; HBeAg qualitative at screening). should be collected only for Part C subjects who are positive), anti-HBe, HBV RNA, hepatitis B core-related antigen (HBcrAg), and HBV DNA.
Resistance surveillance to monitor for the potential development of resistance to the NRTI or HBC34-v35-MLNS-GAALIE will be performed on all subjects receiving study drug. HBV genome sequencing will be performed in subjects with confirmed HBV DNA breakthrough as defined by HBV DNA ≧500 IU/mL measured at two consecutive study visits or early termination of the study with HBV DNA ≧500 IU/mL. Attempt on test subjects. Samples for resistance surveillance will be collected at all study visits noted on the SOA, as it will not be known at the time of the visit if the subject has had a virological breakthrough. Samples taken for resistance surveillance may be used to perform additional viral analysis, including viral sequencing.

Assessment of Immune Responses To examine host immune responses and potential biomarkers of infection, subjects had peripheral immune samples taken with or without liver immune samples (by fine-needle aspiration) at the time points outlined in Figures 15A-16E. may agree to any appropriate sub-study. These appropriate substudies and associated evaluations will be conducted if available at selected sites.

Fc Gamma Receptor (FcγR) Genotyping and Immunoglobulin Allotyping Blood samples for FcγR genotyping and immunoglobulin allotyping were collected at baseline for all subjects in Parts B and C to detect Fc-gamma receptors. The potential association between genetic polymorphisms or immunoglobulin allotypes and antiviral activity of HBC34-v35-MLNS-GAALIE is evaluated.

Statistical methods Statistical analyzes are primarily descriptive. All trial data will be presented by subject data list. For all trial parts, summary tables present results by cohort for HBC34-v35-MLNS-GAALIE and placebo, with placebo subjects aggregated across dose cohorts by route of administration for each part.
This trial will be conducted in accordance with ethical principles that originate from the Declaration of Helsinki and are consistent with Good Clinical Practice (GCP) and applicable regulatory requirements, including record-keeping of essential documentation.

List of abbreviations and definitions of terms in this example ADA Anti-drug antibody AE Adverse event ALT Alanine aminotransferase ANC Absolute neutrophil count AP Alkaline phosphatase AST Aspartate aminotransferase AUC Area under the curve BLQ Below limit of quantitation BMI Body mass index BUN Blood Middle urea nitrogen CLcr Creatinine clearance CRF Case report form CTCAE Common terminology criteria for adverse events DNA Deoxyribonucleic acid ECG Electrocardiogram eCRF Electronic case report form EF End of follow-up ET End of treatment FDA Food and Drug Administration GCP Clinical trial practice standards GGT Gamma glutamyl transferase GLP Good laboratory standards GNA Glycol nucleic acid HBcrAg Hepatitis B core-related antigen HBeAg Hepatitis B e antigen HBIG Hepatitis B immunoglobulin HBsAg Hepatitis B surface antigen HBV Hepatitis B virus HCC Hepatocellular carcinoma HED Human equivalent dose Hgb Hemoglobin ICF Infor Mud consent document ICH International Conference on Harmonization of Pharmaceutical Regulations IgG Immunoglobulin G
IgM immunoglobulin M
IEC Independent Ethics Committee INR International Normalized Ratio IRB Institutional Review Board IV Intravenous IWRS Interactive Web Response System LDH Lactate Dehydrogenase LLN Lower Reference Limit LLOQ Lower Limit of Quantitation LLT Lower term mAb Monoclonal antibody MedDRA Glossary of drug regulatory terms Nab Neutralizing antibody NOAEL No observed adverse effect level OTC OTC drug PK Pharmacokinetics PT Basic terms Q1 1st quartile Q3 3rd quartile RBC Red blood cells (number)
RNA Ribonucleic acid SAD Single dose escalation SAE Serious adverse event SC Subcutaneous SD Standard deviation SoA Evaluation schedule SOC Organ-specific classification SRC Safety review committee SUSAR Unknown and suspected serious side effects TCR Tissue cross-reactivity TEAE Adverse events occurring under treatment US US ULN Upper limit of reference WBC White blood cells (number)
WHO World Health Organization WOCBP Women of childbearing potential [Example 10]

Activation of dendritic cells by HBsAg:HBC34-v35 antibody immune complex ) and the activation of monocyte-derived (mo)DCs in the presence of immune complexes (ICs) formed by HBsAg (supplier: BioIVT) in the serum of HBV+ patients was examined. material and method:
CD14+ monocytes were isolated from human PBMC from healthy donors (n=2) and incubated with RPMI 1640, 10% FBS (Hyclone), 1% non-essential amino acids, 1% glutamine, 1% Pen/Strep, 1% sodium pyruvate. , 50 μM β-mercaptoethanol, 50 ng/mL GM-CSF (Miltenyi) and 1000 U/mL IL-4 (R&D) for 6 days. Differentiated immature monocyte-derived DCs (moDCs) were then treated with HBsAg alone (serum from two patients at 1890 and 4460 IU/ml diluted to a final 250 IU/ml), HBsAg and HBC34-v35-MLNS or HBC34-v35- Stimulation was performed with IC of MLNS_GAALIE (20-100 μg/ml mAb) or mAb alone for 22 hours. The reagent was tested to be endotoxin free. Surface expression of costimulatory markers CD83 and CD86 and HLA-DR was measured by flow cytometry. of ten (10) human proinflammatory cytokines (IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFα). Levels were measured using a Meso Scale Diagnostics (MSD) V-PLEX Proinflammatory Panel 1 Human Kit. Culture medium was used as a negative control. LPS (Sigma, 100ng/ml) served as a positive control.
The data are shown in Figures 20-24B. Immune complexes (ICs) of HBsAg and HBC34-v35-MLNS-GAALIE induced upregulation of costimulatory markers CD83 and CD86, as well as HLA-DR on the surface of moDCs. Furthermore, IC of HBsAg and HBC34-v35-MLNS-GAALIE induced moDCs to secrete the cytokines TNFα, IL-6 and IL-10.
[Example 11]

Reduction of HBsAg in Human Subjects The Phase 1 study described in Example 9 includes the SAD Part A and B cohorts shown in FIG. 25. Extrapolating from preclinical data, an in silico model was generated to predict a reduction in HBsAg of approximately 1.5 log 10 IU/mL after 4 x 60 mg HBC34-v35-MLNS-GAALIE doses (Figure 26).

Figure 27 summarizes certain demographic and baseline characteristics of subjects who received HBC34-v35-MLNS-GAALIE at 6 mg (cohort 1b), 18 mg (cohort 2b), or 75 mg (cohort 3b).

In the Part B SAD cohorts (subjects with chronic HBV with HsAg ≤3000 IU/mL) shown in Figure 25, Cohort 1b (lowest dose cohort 6 mg S.C.), Cohort 2b (18 mg S.C.), and Cohort 3b (75 mg S.C.) each included 2 of the 8 subjects receiving placebo, while the remaining 6 received HBC34-v35-MLNS-GAALIE.

The HBC34-v35-MLNS-GAALIE antibody was well tolerated by the subjects (Figure 28). No clinically significant laboratory abnormalities or changes in liver function tests were observed. No injection site reactions were observed. Subject showed no clinical or laboratory signs of immune complex disease. Two subjects in cohort 1b reported a total of 2 grade 1 adverse events, allergic rhinitis and palpitations without objective tachycardia. The four subjects in Cohort 2b had a total of four adverse events, a grade 1 adverse event of chest discomfort (not of cardiac origin); an abrasion of the subject's right knee, and dizziness; and a grade 2 adverse event of seasonal allergies. reported. Four subjects in Cohort 3b reported a total of eight adverse events. They include three grade 1 adverse events considered to be related to the drug's clinical trials: headache, musculoskeletal stiffness, and nasopharyngitis; four grade 1 adverse events ear infection, fatigue, acne, and sore throat. thirst; and one grade 2 adverse event: headache. No grade transitions or values above the upper limit of normal (ULN) for alanine aminotransferase (ALT) were observed at day 29 post-dosing in cohorts 1b and 2b (Figures 29A-29B), but in participants in cohort 1b. One patient had a grade 1 (ALT) increase 24 weeks after administration.

The 16 subjects in the Part B SAD cohort who received the HBC34-v35-MLNS-GAALIE antibody rapidly achieved >1 log 10 IU/mL reduction in HBsAg within approximately 1 week after administration (Figure 30A). Undetectable or lower than expected free PK of HBC34-v35-MLNS-GAALIE antibody was reported in 2 participants of Cohort 2b, with a reduction in HbsAg of approximately <0.5 log 10 IU/mL (Figure 30A, 30B, 32, and 33B).

Surprisingly, for blinded Cohort 1b, 6 subjects achieved a mean reduction in serum hepatitis B virus surface antigen (HBsAg) of 1.3 log 10 IU/mL by day 8. On day 8, nadir was achieved for most subjects (Figures 30A, 30B, and 31). Notably, these results using 6 mg x 1 exceed predictions from the in silico model using 60 mg x 4 (Figure 26).

Similarly, for blinded cohort 2b, 4 subjects achieved reductions in serum HBsAg of greater than 1.5 log 10 IU/mL by day 8 (Figures 30A, 30B, 31, and 32). Notably, these results using 18 mg x 1 exceed predictions from the in silico model using 60 mg x 4 (see Figure 26). Two subjects who received the 18 mg dose and did not achieve a 1.5 log 10 IU/mL reduction in HbsAg had no detectable HBC34-v35-MLNS-GAALIE antibodies in their samples.

Across the range of baseline HBsAg values, all participants with HBsAg reductions of ≥1 log 10 IU/mL from baseline achieved absolute levels below 30 IU/mL at nadir (Figures 30A, 30B, 31, and 32) . Five of six subjects in Cohort 3b achieved absolute levels below 10 IU/mL at nadir (Figures 30A and 30B). Also, subjects in Cohort 3b achieved maximal (>2 log ₁₀ IU/mL) and most sustained reductions in HBsAg. The average decrease was 1.96 log 10 IU/mL at nadir and 1.5 log 10 IU/mL at day 29 (Figure 31).
[Example 12]

Dose-dependent persistence of hepatitis B surface antigen reduction after single administration of HBC34-v35-MLNS-GAALIE This example demonstrates the safety, tolerability, and tolerability of HBC34-v35-MLNS-GAALIE in participants with chronic HBV infection. Provides data from an ongoing Phase 1 study evaluating tolerability, and antiviral activity.

Methods: This randomized, double-blind, placebo-controlled, phase 1, single-dose, dose-escalation study included adults with hepatitis B antigen (HBeAg)-negative chronic HBV infection without cirrhosis. Participants had HBsAg <3,000 IU/mL at screening and had received nucleoside reverse transcriptase inhibitor (NRTI) therapy for ≥2 months. Eight participants in each cohort were randomized 6:2 to receive a single subcutaneous dose of HBC34-v35-MLNS-GAALIE 6 mg, 18 mg, 75 mg, or 300 mg or placebo. Preliminary data through 8 weeks of follow-up are shown; dose escalation and follow-up are ongoing.

Results: 24 participants were enrolled. Most participants achieved a ≧1 log ₁₀ IU/mL reduction in HBsAg from baseline within 1-3 days of dosing (Figures 30A, 30B, 31, and 32). The greatest and most sustained HBsAg reduction was observed in the 75 mg cohort (Figures 30A, 30B, and 31). For 6 participants per cohort with HBsAg decline >0.2 log ₁₀ IU/mL, mean lows for the 6 mg, 18 mg, and 75 mg groups were 1.30, 1.27, and 1 at nadir, respectively. .96 log ₁₀ IU/mL, and 0.17, 0.20, and 0.82 log ₁₀ IU/mL, respectively, at week 8 (Figure 31). Ten adverse events were reported, all of grade 1 or 2 severity (Figure 28). No clinically significant laboratory abnormalities or signs of immune complex disease were observed. Data from participants who received a single dose of 300 mg HBC34-v35-MLNS-GAALIE are shown in Figures 30A and 30B.

Conclusion: Single administration of 6 mg, 18 mg, or 75 mg HBC34-v35-MLNS-GAALIE showed a rapid reduction in HBsAg. HBC34-v35-MLNS-GAALIE was generally safe and well tolerated. These data support the use of HBC34-v35-MLNS-GAALIE for functional cure of patients with chronic HBV infection.
[Example 13]

Measurement of PK in Human Subjects The purpose of this study is to characterize the serum pharmacokinetics (PK) of HBC34-v35-MLNS-GAALIE in subjects of SAD B Cohort, SAD C Cohort 1c.

The concentration of HBC34-v35-MLNS-GAALIE in serum was determined using a validated electrochemiluminescence method on a Meso Scale Discovery (Rockville, MD) platform with an LLOQ of 10 ng/mL. PK parameters were estimated using standard noncompartmental methods in WinNonlin®, V8.2 (Certara LP, Princeton, NJ) and summarized using descriptive statistics. Bioavailability (F%) was calculated by the formula BA=AUC _sc /AUC _iv ×100, where AUC _iv and AUC _sc represent the area under the curve after SC administration.

Free and total HBC34-v35-MLNS-GAALIE in all 6 active participants after a single 75 mg dose in cohort 3b aligns with corresponding >1 log HBsAg reduction sustained for at least 28 days (Figure 33C and Fig. 35C). The total PK profiles of Cohorts 1b and 3b approaches project PK profiles based on data from healthy subjects (Figures 35A, 35B, 36, 39, and 40). Unexpected free HBC34-v35-MLNS-GAALIE PK was observed in 7 of 12 active subjects after 18 mg administration in cohorts 2b and 1c. Furthermore, low antiviral activity (<0.5 log HBsAg) was observed in 4 of 7 subjects who showed an unexpected PK profile. 8/24 HBV showed unexpected free PK profiles at 6 mg (1b n=1/6; Figures 33A and 35A) and 18 mg (2b n=3/6; 1c n=4/6; Figures 33B and 35B) Total PK in participants was aligned with free PK.
[Example 14]

Pharmacokinetics in Subjects and Safety Measurements in Healthy Human Subjects Forty-one healthy human subjects (n=8 per cohort, plus 1 subject replaced for non-safety-related reasons) were enrolled and treated with HBC34-v35. - Received MLNS-GAALIE or placebo (Figure 37). Cohorts received a single dose of either 90 mg (s.c.), 300 mg (s.c.), 900 mg (s.c.), 900 mg (intravenous), or 3,000 mg (intravenous). HBC34-v35-MLNS-GAALIE was absorbed after subcutaneous injection with a median T _max of 7 days. Exposure (C _max and AUC) increased dose-proportionally over the 90-900 mg subcutaneous range (Figure 39). Intersubject variability within each cohort was generally low (CV approximately 35%). The PK profile of HBC34-v35-MLNS-GAALIE is consistent with a typical IgG, with a half-life of approximately 28 days. HBC34-v35-MLNS-GAALIE was well tolerated across doses from 90 to 3,000 mg (Figure 38). A total of 24/41 (59%) subjects experienced adverse events, the majority being grade 1 (Figure 38). No serious adverse events were reported and no clinically significant effects on laboratory parameters or electrocardiograms were observed. These results demonstrate that HBC34-v35-MLNS-GAALIE is safe and well-tolerated in healthy human subjects after single doses up to 3,000 mg, with favorable PK properties supporting subcutaneous administration. It is shown that.
[Example 15]

Pharmacokinetics and Safety Measurements in Healthy Human Subjects The purpose of this study was to evaluate the safety and tolerability of HBC34-v35-MLNS-GAALIE in healthy subjects and to characterize the serum pharmacokinetics (PK). there were.

Part A is a randomized, blinded, placebo-controlled study of HBC34-v35-MLNS-GAALIE in healthy subjects aged 18-55 years with creatinine clearance <90 mL/min. Figure 37 summarizes certain demographic characteristics of the subjects and the dose of HBC34-v35-MLNS-GAALIE administered to the subjects. Eight subjects per cohort were randomized 6:2 to receive a single dose of HBC34-v35-MLNS-GAALIE or placebo by subcutaneous (SC) or intravenous (IV) injection. Serum PK samples were collected over 24 hours (day 1) and on days 3, 7, 14, and weeks 4, 8, 12, 18, and 24.

The concentration of HBC34-v35-MLNS-GAALIE in serum was determined using a validated electrochemiluminescence method on a Meso Scale Discovery (Rockville, MD) platform with an LLOQ of 10 ng/mL. PK parameters were estimated using standard noncompartmental methods in WinNonlin®, V8.2 (Certara LP, Princeton, NJ) and summarized using descriptive statistics. Bioavailability (F%) was calculated by the formula BA=AUC _sc /AUC _iv ×100, where AUC _iv and AUC _sc represent the area under the curve after IV and SC administration, respectively. Adverse event (AE) monitoring, clinical and physical examinations, and electrocardiographic evaluation were performed throughout the study. Injection site tolerability evaluations were performed at approximately 30 minutes, 2, 12, 24, and 48 hours, and one week after administration.

Adverse events are summarized in Figure 38. The most commonly reported AE across all groups was headache, observed in 10/41 (24.4%) participants. Injection site reactions were reported in 6/41 (14.6%) participants, all of grade 1 severity except injection site erythema, a grade 2 AE, which resolved without intervention. No gray 3/4 AEs, SAEs, or AEs leading to study discontinuation were observed. No clinically significant laboratory abnormalities were observed.

HBC34-v35-MLNS-GAALIE was absorbed after SC injection with a median T _max of 3-7 days and a preliminary half-life t _1/2 of approximately 25 days. Dose-proportional increases in C _max and AUC _inf were observed over the SC dose range of 90-900 mg. Intersubject variability within each cohort was generally low (CV approximately 35%) for all PK parameters. The bioavailability of HBC34-v35-MLNS-GAALIE after SC administration was approximately 76%. The PK data is summarized in Figures 39 and 40.

Therefore, HBC34-v35-MLNS-GAALIE was well tolerated in healthy volunteers after single doses up to 3000 mg. Adverse events were generally mild, and no adverse events led to study discontinuation. Systemic exposure of HBC34-v35-MLNS-GAALIE was maintained at the dose range evaluated for 24 weeks. The bioavailability and half-life of HBC34-v35-MLNS-GAALIE after SC administration are estimated to be 76% and 25 days, respectively.
[Example 16]

Phase 1 clinical trial, Part D
Part D is chronic HBV without cirrhosis who is not on nucleoside reverse transcriptase inhibitor therapy, has HBV DNA ≥1,000 IU/mL, is HBeAg negative or HBeAg positive, and has any HBsAg level. A randomized, double-blind, placebo-controlled, single-dose, dose-escalation study of HBC34-v35-MLNS-GAALIE administered to infected adult subjects. Part D consisted of three arbitrary cohorts, each of 8 participants, who were randomized 6:2 to receive HBC34-v35-MLNS-GAALIE or placebo on study day 1. be done. Each cohort can evaluate HBC34-v35-MLNS-GAALIE doses up to 900 mg administered by subcutaneous injection. Subjects will be required to attend the study site until week 8 for evaluations including, but not limited to, physical examination, vital signs, laboratory tests, pharmacokinetic evaluation, efficacy evaluation, and review of adverse events and concomitant medications. will return to. Subjects meeting certain criteria at week 8 may be followed up to week 40.

Table of sequences and sequence numbers (sequence listing):

U.S. Provisional Application No. 63/141,915 filed on January 26, 2021; U.S. Provisional Application No. 63/142,779 filed on January 28, 2021; and United States Provisional Application No. 63/142,779 filed on June 11, 2021. U.S. Provisional Application No. 63/209,875, filed on October 14, 2021, and U.S. Provisional Application No. 63, filed on November 18, 2021. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to herein or in the attached application data sheets, including No. 280,971, are incorporated herein by reference. is incorporated herein by reference in its entirety to the extent consistent with .

From the foregoing, it will be understood that while particular embodiments of the invention are described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims (55)

91. A method of treating hepatitis B virus (HBV) infection in a subject, the method comprising administering to the subject a single dose of a pharmaceutical composition comprising an antibody, wherein the antibody is chain amino acid sequence and the light chain amino acid sequence of SEQ ID NO: 93;
(a) said single dose pharmaceutical composition comprises at least 6 mg of said antibody;
(b) after administration of the single dose, the subject's serum HBsAg decreases by at least 1.0 log ¹⁰ IU/mL, 1.5 log ¹⁰ IU/mL, or more compared to baseline; (c) The method wherein the reduction in serum HBsAg in said subject persists for 1, 2, 3, 4, 5, 6, 7, 8, or more days after administration of said single dose. 91. A method of treating hepatitis B virus (HBV) infection in a subject, the method comprising administering to the subject a single dose of a pharmaceutical composition comprising an antibody, wherein the antibody is chain amino acid sequence and the light chain amino acid sequence of SEQ ID NO: 93;
(a) said single dose pharmaceutical composition comprises at least 75 mg of said antibody;
(b) after administration of the single dose, the subject's serum HBsAg decreases by at least 1.0 log ¹⁰ IU/mL, at least 1.5 log ¹⁰ IU/mL, or more compared to baseline;
(c) said subject's serum HBsAg decreases at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 after administration of said single dose; A method lasting for 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 days or more. A method of treating hepatitis B virus (HBV) infection in a subject, said method comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody;
(a) the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 91 and a light chain amino acid sequence of SEQ ID NO: 93;
(b) at least 1000 ng/mL of the antibody remains unbound to serum HBsAg for at least 14 days after administration of said single dose;
(c) the subject has a baseline serum HBsAg level of less than 3000 IU/mL. 4. The method of any one of claims 1-3, wherein the subject's serum HBsAg decreases by at least 1.0 log ¹⁰ IU/mL within 8 days of administration of a single dose compared to baseline. said single dose of the pharmaceutical composition comprises at least 75 mg of said antibody, and said subject's serum HBsAg is at least 1.5 log ¹⁰ IU/dose within 8 days of administration of said single dose as compared to baseline. 5. The method according to any one of claims 1 to 4, wherein the mL is reduced. The method of any one of claims 1-5, wherein the subject's serum HBsAg is reduced by at least 0.5 log ¹⁰ IU/mL on day 56 after administration of the single dose compared to baseline. . 7. The method of any one of claims 1-6, wherein the subject has a C _max of the antibody between 300 ng/mL and 6,000 ng/mL. C _max of the concentration of the antibody in the subject is at least 300ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL, 1000ng/mL, 1100ng/mL, 1200ng / Ml, 1300 ng / ml, 1400 ng / ml, 1500 ng / ml, 1600 ng / ml, 1700 ng / ml, 1800 ng / ml, 1700 ng / ml, 2000 ng / ml, 2000 ng / ml, 2100 ng / ml, 2200 ng / ml , 2300 ng / ml, 2400 ng / ml , 2500ng/mL, 2600ng/mL, 2700ng/mL, 2800ng/mL, 2900ng/mL, 3000ng/mL, 3100ng/mL, 3200ng/mL, 3300ng/mL, 3400ng/mL, 3500ng/mL, 3600ng/mL, 37 00ng / Ml, 3800 ng / ml, 3900 ng / ml, 4000 ng / ml, 4100 ng / ml, 4200 ng / ml, 4300 ng / ml, 4400 ng / ml, 4500 ng / ml, 4600 ng / ml, 470 0 ng / ml, 4800 ng / ml, 4900 ng / ml , 5000ng/mL, 5100ng/mL, 5200ng/mL, 5300ng/mL, 5400ng/mL, 5500ng/mL, 5600ng/mL, 5700ng/mL, 5800ng/mL, or 5900ng/mL, according to claim 7. Method. The single dose of the pharmaceutical composition may be up to 10 mg, up to 15 mg, up to 18 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 mg, up to 75 mg, up to 90 mg, up to 300 mg. up to 900 mg, or up to 3000 mg of said antibody,
or said single dose of the pharmaceutical composition is in the range 6 mg to 3000 mg, or in the range 10 mg to 3000 mg, or in the range 25 mg to 3000 mg, in the range 30 mg to 3000 mg, in the range 50 mg to 3000 mg, in the range 60 mg to 3000 mg, in the range 75 mg ~3000mg range, 90mg~3000mg range, 100mg~3000mg range, 150mg~3000mg range, 200mg~3000mg range, 300mg~3000mg range, 500mg~3000mg range, 750mg~3000mg range, 900mg~3000 mg 1500 mg to 3000 mg, 2000 mg to 3000 mg,
or said single dose of the pharmaceutical composition is in the range 6 mg to 900 mg, or in the range 10 mg to 900 mg, or in the range 25 mg to 900 mg, in the range 30 mg to 900 mg, in the range 50 mg to 900 mg, in the range 60 mg to 900 mg, in the range 75 mg. 900mg to 900mg, 90mg to 900mg, 100mg to 900mg, 150mg to 900mg, 200mg to 900mg, 300mg to 900mg, 500mg to 900mg, 750mg to 900mg. Contains antibodies;
or said single dose of the pharmaceutical composition is in the range 6 mg to 500 mg, or in the range 10 mg to 500 mg, or in the range 25 mg to 500 mg, in the range 30 mg to 500 mg, in the range 50 mg to 500 mg, in the range 60 mg to 500 mg, in the range 75 mg. 500 mg, 90 mg to 500 mg, 100 mg to 500 mg, 150 mg to 500 mg, 200 mg to 500 mg, 300 mg to 500 mg, 400 mg to 500 mg,
or said single dose of the pharmaceutical composition is in the range 6 mg to 300 mg, or in the range 10 mg to 300 mg, or in the range 25 mg to 300 mg, in the range 30 mg to 300 mg, in the range 50 mg to 300 mg, in the range 60 mg to 300 mg, in the range 75 mg. 300 mg, 90 mg to 300 mg, 100 mg to 300 mg, 150 mg to 300 mg, 200 mg to 300 mg,
or wherein said single dose of the pharmaceutical composition is in the range 6 mg to 200 mg, or in the range 10 mg to 200 mg, or in the range 25 mg to 200 mg, in the range 30 mg to 200 mg, in the range 50 mg to 200 mg, in the range 60 mg to 200 mg, in the range 75 mg. 200 mg, 90 mg to 200 mg, 100 mg to 200 mg, 150 mg to 200 mg,
or said single dose of the pharmaceutical composition is in the range 6 mg to 100 mg, or in the range 10 mg to 100 mg, or in the range 25 mg to 100 mg, in the range 30 mg to 100 mg, in the range 50 mg to 100 mg, in the range 60 mg to 100 mg, in the range 75 mg. -100 mg, 75 mg to 100 mg, 90 mg to 100 mg,
or said single dose pharmaceutical composition comprises said antibody in an amount in the range 6 mg to 25 mg, or in the range 10 mg to 25 mg, or in the range 15 mg to 25 mg, or in the range 20 mg to 25 mg;
or said single dose of the pharmaceutical composition is in the range 6 mg to 50 mg, or in the range 6 mg to 25 mg, or in the range 6 mg to 50 mg, or in the range 10 to 50 mg, or in the range 10 to 25 mg, or in the range 6 mg to 15 mg. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130 , 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255 , 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380 , 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505 , 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630 , 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755 , 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880 , 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 mg, or more of the above-mentioned antibodies,
or the single dose of the pharmaceutical composition is less than 3000 mg, less than 2500 mg, less than 2000 mg, less than 1500 mg, less than 1000 mg, less than 900 mg, less than 500 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 90 mg, less than 75 mg, less than 50 mg, 9. A method according to any one of claims 1 to 8, comprising said antibody in an amount of less than 25 mg, or less than 10 mg. The single dose of the pharmaceutical composition may be in the range of 100 mg/mL to 200 mg/mL, such as 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg. 10. A method according to any one of claims 1 to 9, comprising the antibody at a concentration of 180 mg/mL, 180 mg/mL, 190 mg/mL or 200 mg/mL, preferably 150 mg/mL. 11. The method of any one of claims 1-10, wherein the single dose of the pharmaceutical composition comprises about 75 mg of the antibody. 11. The method of any one of claims 1 to 10, wherein the single dose of the pharmaceutical composition comprises about 90 mg of the antibody. 11. The method of any one of claims 1 to 10, wherein the single dose of the pharmaceutical composition comprises up to 300 mg of the antibody. 11. The method of any one of claims 1 to 10, wherein the single dose of the pharmaceutical composition comprises up to 900 mg of the antibody. 11. The method of any one of claims 1 to 10, wherein the single dose of the pharmaceutical composition comprises up to 3,000 mg of the antibody. The method comprises administering the single dose by subcutaneous injection, and the single dose may comprise or consist of 6 mg of the antibody, 18 mg of the antibody, or 75 mg of the antibody. , the method according to any one of claims 1 to 15. 17. The method of any one of claims 1 to 16, comprising administering said single dose by intravenous injection. 18. A method according to any one of claims 1 to 17, wherein the pharmaceutical composition further comprises water, optionally water. 19. A method according to any one of claims 1 to 18, wherein the pharmaceutical composition comprises histidine, optionally at a concentration in the range 10mM to 40mM, such as 20mM. 19. The pharmaceutical composition further comprises a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8% or 9%, preferably about 7% (w/v). The method described in any one of the above. The pharmaceutical composition further comprises a surfactant or a triblock copolymer, optionally a polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), wherein the polysorbate or poloxamer 188 is optionally comprised between 0.01% and 0.05%. % (w/v), preferably 0.02% (w/v). The pharmaceutical composition has a pH in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or in the range of 5.8, 5.9, 6.0, 6.1, or 6.2. 22. A method according to any one of claims 1 to 21, comprising: The pharmaceutical composition comprises:
(i) 150 mg/mL of said antibody;
(ii) USP Water;
(iii) 20mM histidine;
(iv) 7% sucrose, and (v) 0.02% PS80.
23. The method of claim 22, wherein the method comprises: and has a pH of 6. 24. The method according to any one of claims 1 to 23, wherein the subject is an adult. 25. The method of claim 24, wherein the subject ranges in age from 18 to 65 years. 26. The method according to any one of claims 1 to 25, wherein the subject's body weight is between 40 kg and 125 kg and/or the subject has a body mass index (BMI) between 18 and 35 kg/ ^m2 . 1 . The subject has chronic HBV infection, e.g. as defined by positive sera for HBsAg, HBV DNA, and/or HBeAg on two occasions, the two occasions being at least 6 months apart. 1 . 27. The method according to any one of 26 to 26. 28. The method of any one of claims 1-27, wherein the subject does not have cirrhosis. The absence of cirrhosis
Fibroscan evaluation (e.g. within 6 months prior to administration of said single dose of pharmaceutical composition) or liver biopsy (e.g. within 12 months prior to administration of said single dose pharmaceutical composition)
29. The method according to claim 28, wherein the absence of cirrhosis is determined by the absence of Metavir F3 fibrosis or the absence of F4 cirrhosis. Claim 1, wherein said subject has received a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI), optionally within 120 days, and optionally within 60 days, before said single dose is administered. 29. The method according to any one of 29 to 29. the NRTI comprises one or more of tenofovir, tenofovir disoproxil (e.g., tenofovir disoproxil fumarate), tenofovir alafenamide, entecavir, lamivudine, adefovir, and adefovir dipivoxil; 31. The method of claim 30. 32. The method of any one of claims 1-31, wherein the subject has a serum HBV DNA concentration of less than 100 IU/mL for a period of no more than 28 days before the single dose is administered. said subject has a serum HBsAg concentration of less than 3,000 IU/mL before said single dose is administered and has a serum HBsAg concentration of less than 1,000 IU/mL before said single dose is administered; 33. A method according to any one of claims 1 to 32. said subject has a serum HBsAg concentration of 3,000 IU/mL or more for a period of no more than 28 days before said single dose is administered, and for a period of no more than 28 days before said single dose is administered; 33. The method according to any one of claims 1 to 32, which may have a serum HBsAg concentration of 1,000 IU/mL or more. 35. The method of any one of claims 1-34, wherein the subject has been HBe antigen (HBeAg) negative for no more than 28 days before the single dose is administered. 36. The method of any one of claims 1-35, wherein the subject was negative for anti-HB antibodies for no more than 28 days before the single dose was administered. before said subject is administered said single dose;
(i) does not have fibrosis and/or does not have cirrhosis; and/or (ii) has an alanine aminotransferase (ALT) lower than two times the upper limit of normal (ULN);
37. A method according to any one of claims 1 to 36. On day 56 after administration of said single dose, said subject has reduced serum HBsAg to less than half of said subject's serum HBsAg from day 0 to day 28 prior to administration of said single dose. 38. The method of any one of claims 1 to 37, wherein the method has a decrease in the concentration of HBsAg in serum (e.g. the concentration of HBsAg determined using the Abbott ARCHITECT assay). After administration of said single dose (e.g., on day 56 after administration of said single dose), said subject:
(i) has reduced or less severe intrahepatic spread of HBV compared to a reference subject, and/or (ii) comprises an adaptive immune response against HBV;
39. A method according to any one of claims 1 to 38. 40. A method according to any one of claims 1 to 39, wherein the subject is male. 40. A method according to any one of claims 1 to 39, wherein the subject is female. A pharmaceutical composition comprising an antibody, wherein the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 91 and a light chain amino acid sequence of SEQ ID NO: 93, wherein the pharmaceutical composition comprises an antibody in the range of 100 mg/mL to 200 mg/mL; For example, 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, or 200 mg/mL, preferably 150 mg/mL. mL, and immediately after administering said composition to a subject in need thereof, at least 1000 ng/mL of the antibody remains unbound to serum HBsAg for at least 14 days after administration of said single dose. , wherein said subject has a baseline serum HBsAg level of less than 3000 IU/mL. 43. The pharmaceutical composition of claim 42, comprising up to 6 mg, up to 18 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody. 44. A pharmaceutical composition according to claim 42 or 43, comprising about 75 mg of said antibody. 44. A pharmaceutical composition according to claim 42 or 43, comprising about 90 mg of said antibody. 44. A pharmaceutical composition according to claim 42 or 43, comprising about 300 mg of said antibody. 44. A pharmaceutical composition according to claim 42 or 43, comprising about 900 mg of said antibody. 44. A pharmaceutical composition according to claim 42 or 43, comprising about 3,000 mg of said antibody. 49. A pharmaceutical composition according to any one of claims 42 to 48, further comprising water, optionally USP water. A pharmaceutical composition according to any one of claims 42 to 49, further comprising histidine in the pharmaceutical composition, optionally at a concentration of 10mM to 40mM, such as 20mM. 51. According to any one of claims 42 to 50, further comprising a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8% or 9%, preferably about 7% (w/v). Pharmaceutical compositions as described. It further comprises a surfactant which may be a polysorbate, preferably polysorbate 80 (SP80), wherein said polysorbate is optionally in the range of 0.01% to 0.05% (w/v), preferably 0.02% (w/v). /v) according to any one of claims 42 to 51. from claim 42, having a pH in the range 5.8 to 6.2, in the range 5.9 to 6.1, or in the range 5.8, 5.9, 6.0, 6.1, or 6.2. 53. The pharmaceutical composition according to any one of 52. (i) 150 mg/mL of said antibody;
(ii) USP Water;
(iii) 20mM histidine;
(iv) 7% sucrose, and (v) 0.02% PS80.
54. A pharmaceutical composition according to any one of claims 42 to 53, comprising: and having a pH of 6. 42. The method according to any one of claims 1 to 41, wherein the subject is HBeAg negative or HBeAg positive.